Pest resistant plants

ABSTRACT

The disclosure provides an isolated nucleic acid molecule encoding a 7-epizingiberene synthase, a chimeric gene comprising said nucleic acid molecule, vectors comprising the same, as well as isolated 7-epizingiberene synthase proteins themselves. In addition, transgenic plants and plant cells comprising a gene encoding a 7-epizingiberene synthase, optionally integrated in its genome, and methods for making such plants and cells, are provided. Especially Solanaceae plants and plant parts (seeds, fruit, leaves, etc.) with enhanced insect pest resistance are provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/571,095, filed Dec. 15, 2014; which is a Continuation of U.S.application Ser. No. 14/122,579, filed Jan. 28, 2014; which is theNational Phase of International Patent Application No.PCT/NL2012/050382, filed May 31, 2012; which claims priority to U.S.Provisional Application Nos. 61/491,339, filed May 31, 2011 and61/607,008, filed Mar. 6, 2012. The contents of these applications areherein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to an isolated nucleic acid moleculeencoding a 7-epizingiberene synthase, a chimeric gene comprising saidnucleic acid molecule, vectors comprising the same, host cellscomprising such vector, as well as isolated zingiberene synthaseproteins themselves. The present invention further provides a method forpreparing 7-epizingiberene using such nucleic acid molecule. Inaddition, transgenic plants and plant cells comprising a gene encodingsuch zingiberene synthase, optionally integrated in its genome, andmethods for making such plants and cells, are provided. EspeciallySolanaceae plants and plant parts (seeds, fruit, leaves, etc.) withenhanced insect pest resistance are provided.

BACKGROUND OF THE INVENTION

Some common insect pests of agronomically important crop plants such astomatoes include the South American Tomato Leaf Miner (Tuta absoluta),stink bugs, cutworms, hornworms, aphids, cabbage loopers, whiteflies(Bemisia and Trialeurodes), fruitworms, flea beetles, spider mites suchas Tetranychus urticae (the glass house red spider mite), Panonychusulmi (fruit tree red spider mite) and Panonychus citri (citrus redmite), insects of the order Diptera, and Colorado potato beetles(Leptinotarsa decemlineata).

For example, whiteflies of the genera Bemisia (sweet potato whitefly)and Trialeurodes (greenhouse whitefly) are major pests of crop plantsthroughout the world, causing economic losses especially due to thetransmission of plant viruses during feeding (i.e. they act as ‘virusvectors’). Bemisia tabaci is capable of transmitting more than 60different members of the Geminiviridae, many of which belong to theBegomoviruses such as African cassava mosaic virus (ACMV), Bean goldenmosaic virus (BGMV), Bean dwarf mosaic virus, Tomato yellow leaf curlvirus (TYLCV), Tomato mottle virus (ToMoV), and others, plus a number ofcriniviruses. Both tropical and temperate crops are affected, such astomatoes, beans, cucurbits, potatoes, cotton, cassava and sweetpotatoes.

To date, the main control strategy for insect pests is the applicationof insecticides, aimed at killing adults, juveniles and eggs. Besidesthe substantial costs of insecticide application this practice has asevere environmental impact. Moreover, many insect pests are difficultto control with insecticides due to emerging resistance to the activeingredients.

In order to reduce insecticide application, there is a need for new waysof controlling crop damage and losses due to plant insect pests, both infield-grown and greenhouse-grown crops. From literature it is known thatvolatile components can directly influence insect behaviour (e.g. Bruceet al., 2005, Trends Plant Sci. 10: 269-74). One way to control virustransmission by plant insect pests is by identifying insect repellents,which can be applied on or near the crop plants or can be produced inthe crop.

EP 0 583 774 describes the use of vegetable oil to reduce phytotoxicityof foliar insect control agents, whereby any type of insect controlagent may be used.

Glandular trichomes are prominent on foliage and stems of the genusLycopersicon (now classified as Solanum) and have been shown to producea large number of secondary compounds, such as mono- and sesquiterpenehydrocarbons, sesquiterpene acids, methylketones and sugar esters.Several studies have tried to correlate the density of glandulartrichomes with resistance against plant pests, such as maize earworm(Heliothis zea) or Colorado potato beetle (Kauffman and Kennedy, 1989, JChem Ecol 15, 1919-1930; Antonious, 2001, J Environ Sci Health B 36,835-848 and Antonious et al. 2005, J Environ Sci Health B 40: 619-631).Also the methylketones 2-undecanone and 2-tridecanone, stored in theglandular trichomes of L. hirsutum f. glabratum (renamed to S.habrochaites) were shown to exhibit a toxic effect against fourth instarlarvae of Colorado potato beetle and adult whiteflies B. tabaci,respectively (Antonious et al. 2005, J Environ Sci Health B 40:619-631).

Antonious and Kochhar (J Environm Science and Health B, 2003, B38:489-500) extracted and quantified zingiberene and curcumene from wildtomato accessions with the goal of selecting wild tomato accessions thatcan be used for the production of sesquiterpene hydrocarbons for naturalinsecticide production. However, whether such compounds are able to beused as whitefly repellents or attractants was not disclosed. It ismentioned that zingiberene has been associated with Colorado beetleresistance and beet armyworm resistance, while curcumene has beenassociated with insecticidal effects. The wild tomato species L.hirsutum f. typicum (S. habrochaites) is mentioned to be resistant to B.argentifolii (now named B. tabaci) (Heinz et al. 1995, 88:1494-1502),but trichome based plant resistance could, of course, have variouscauses and from this paper one cannot make inferences regarding thepresence or identity of compounds which have properties for attractingor repelling whiteflies.

Freitas et al. (Euphytica 2002, 127: 275-287) studied the geneticinheritance of the genes for the production of both the sesquiterpenezingiberene and glandular trichome types I, IV, VI and VII ininterspecific crosses between L. esculentum (S. lycopersicum; cultivatedtomato, no zingiberene) and wild L. hirsutum var. hirsutum (S.habrochaites; high in zingiberene). Zingiberene content in F₂ plantscontributed to B. argentifolii (B. tabaci) resistance by correlation andit was suggested to breed plants with simultaneously high levels ofzingiberene, 2-tridecanone and/or acylsugars to contribute to higherlevels of whitefly resistance.

ES 2341085 discloses exogenous application of alpha-zingiberene as arepellent and insecticide against T. absoluta and other insects thataffect tomato crops. Alpha-zingiberene may be applied in its pure form,or in its natural form through the use of essential oils containing themolecule in appropriate concentrations.

De Azavedo et al., Euphytica 2003, 134, 247-351 describe the effect ofendogenous zingiberene mediated resistance to T. absoluta.

According to Pushkar, N. K. and Balawant, S. J. (2001) “Alternativemedicine: Herbal drugs and their critical appraisal” in Jucker, E.Progress in Drug Research, Vol. 57, ginger essential oil containsalpha-zingiberene, but not 7-epizingiberene (Table 4, page 46).

Bleeker et al., Phytochemistry 2011, 72(1):68-73 disclose that7-epizingiberene and R-curcumene, both purified from Solanumhabrochaites (PI127826), act as repellent to Bemisia tabaci whiteflies,while stereoisomers alpha-zingiberene and S-curcumene from Zingiberofficinalis oil (ginger oil) do not. Bio-assays showed that a cultivatedtomato could be made less attractive to B. tabaci than its neighbouringsiblings by the addition of the tomato stereoisomer 7-epizingiberene orits derivative R-curcumene (abstract).

Davidovich-Rikanati et al., The Plant Journal 2008, 56(2):228-238disclose the transformation of tomato plants with a construct harbouringthe alpha-zingiberene synthase of lemon basil (Ocimum basilicum L.)coupled to the fruit ripening-specific tomato polygalacturonase promoter(PG). The overexpression of alpha-zingiberene synthase results in theproduction of alpha-zingiberene by the transgenic tomatoes. It isfurther described that alpha-zingiberene is a major leaf oilsesquiterpene in Solanum hirsutum, and this trait has been associatedwith resistance to B. tabaci.

Iijima et al., Plant Physiology 2004, 136(3):3724-3736 disclose theisolation and expression in E. coli of an alpha-zingiberene synthase ofsweet basil.

Although several methods exist for combating plant insect pests, thereis still a need for adequate protection against insect pests such as,for example, a tabaci.

SUMMARY OF THE INVENTION

The present inventors have now identified a gene encoding a7-epizingiberene synthase protein from Solanum habrochaites (ShZIS).

Thus, in a first aspect, the present invention provides an isolatedprotein comprising the amino acid sequence of SEQ ID NO: 1 or an aminoacid sequence comprising at least 92% amino acid sequence identity tothe amino acid sequence of SEQ ID NO:1 over the entire length. Thepresent invention further provides an isolated, synthetic or recombinantnucleic acid sequence selected from the group comprising: a) a nucleicacid sequence of SEQ ID NO: 2; b) a nucleic acid sequence that encodes apolypeptide comprising an amino acid sequence of SEQ ID NO: 1; c) anucleic acid sequence that is at least 92% identical to the nucleic acidsequences of (a) or (b), and encodes a 7-epizingiberene synthase; d) anucleic acid sequence encoding a polypeptide comprising an amino acidsequence of SEQ ID NO:1 wherein at least one amino acid is substituted,deleted, inserted or added and wherein the polypeptide is functionallyequivalent to the polypeptide consisting of the amino acid sequence ofSEQ ID NO:1; and e) a nucleic acid sequence that hybridizes understringent conditions to the nucleic acid sequences of (a), (b), or (c),and encodes a 7-epizingiberene synthase; a chimeric gene comprising apromoter, optionally active in plant cells, operably linked to suchnucleic acid molecule, and, optionally, further operably linked to a 3′untranslated nucleic acid molecule, as well as a vector comprising suchchimeric gene. A host cell comprising such vector is also included inthe present invention.

The present invention is also directed to a method for preparing7-epizingiberene and/or R-curcumene comprising the steps of: a)transforming a host cell with the nucleic acid molecule, a chimeric geneor a vector according to the present invention; b) culturing said hostcell under conditions permitting production of 7-epizingiberene; c)optionally, isolating the 7-epizingiberene produced in step b); and d)optionally, dehydrogenating said 7-epizingiberene to produceR-curcumene.

In another aspect, the present invention is concerned with a method forproducing 7-epizingiberene from zFPP in a host cell, comprising: a)introducing into said host cell a first nucleic acid sequence encoding azFPS as shown in SEQ ID NO:6 or an amino acid sequence comprising atleast 80% amino acid sequence identity to the amino acid sequence of SEQID NO:6 over the entire length, and a second nucleic acid sequenceencoding a 7-epizingiberene synthase comprising the amino acid sequenceof SEQ ID NO: 1 or an amino acid sequence comprising at least 92% aminoacid sequence identity to the amino acid sequence of SEQ ID NO:1; b)culturing the transformed host cell in suitable conditions for theexpression of said first and said second nucleic acid sequences; and, c)optionally, collecting the zFPP and/or the 7-epizingiberene contained insaid host cell and/or in the culture medium.

In a further aspect, the present invention pertains to a transgenicplant, plant cell, seed or fruit, comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:1 or an amino acidsequence comprising at least 92% amino acid sequence identity to theamino acid sequence of SEQ ID NO:1 over the entire length.

In yet another aspect, the present invention relates to a Solanumlycopersicum plant, plant cell, seed or fruit comprising a nucleic acidsequence encoding the amino acid sequence of SEQ ID NO:1 or an aminoacid sequence comprising at least 92% amino acid sequence identity tothe amino acid sequence of SEQ ID NO:1 over the entire length.Preferably, said Solanum lycopersicum plant, plant cell, seed or fruitfurther comprises a nucleic acid sequence encoding aZ,Z-farnesyl-diphosphate synthase (herein also referred to as a “zFPP”or Z,Z-FPP).

In a further aspect, the present invention provides a method forproducing a transgenic plant having enhanced insect pest resistancecompared to a non-transgenic control plant, said method comprising thesteps of: (a) transforming a plant or plant cell with a nucleic acidmolecule encoding the amino acid sequence of SEQ ID NO: 1 or an aminoacid sequence comprising at least 92% amino acid sequence identity tothe amino acid sequence of SEQ ID NO:1 over the entire length, operablylinked to a promoter active in plant cells, and (b) regenerating aplant. Said nucleic acid molecule may be integrated into the genome ofsaid plant. Said method may further comprise the step of (c) screeningthe regenerated plant, or a plant derived therefrom by selfing orcrossing, for resistance to one or more insect pests and identifying aplant comprising enhanced resistance to one or more of said insectpests. The promoter may be an insect pest inducible promoter. The plantmay belong to the family Solanaceae. The plant may be of the genusSolanum.

In an embodiment, the method further comprises the step of transformingthe plant or plant cell with a nucleic acid molecule encoding the aminoacid sequence of SEQ ID NO:5 or an amino acid sequence comprising atleast 80% amino acid sequence identity to the amino acid sequence of SEQID NO:6, preferably over the entire length, operably linked to apromoter active in plant cells.

In a further aspect, the present invention relates to the use of anucleic acid molecule encoding the amino acid sequence of SEQ ID NO:1 oran amino acid sequence comprising at least 92% amino acid sequenceidentity to the amino acid sequence of SEQ ID NO:1 over the entirelength for the generation of insect pest resistant plants.

In a final aspect, the present invention is concerned with a method foridentifying a genomic polymorphism between Solanum habrochaites andspecies of the Solanum type that are sexually compatible with Solanumhabrochaites comprising detecting a genomic polymorphism with molecularmarkers comprising all or part of the gene encoding the amino acidsequence of SEQ ID NO:1 or an amino acid sequence having at least 92%sequence identity with the amino acid sequence of SEQ ID NO:1 so as tocontrol the introgression of the corresponding gene in said species.

GENERAL DEFINITIONS

The term “nucleic acid molecule” (or “nucleic acid sequence”) refers toa DNA or RNA molecule in single or double stranded form, particularly aDNA encoding a protein according to the invention. An “isolated nucleicacid sequence” refers to a nucleic acid sequence which is no longer inthe natural environment from which it was isolated, e.g. the nucleicacid sequence in a bacterial host cell or in the plant nuclear orplastid genome.

The terms “protein” or “polypeptide” are used interchangeably and referto molecules consisting of a chain of amino acids, without reference toa specific mode of action, size, 3 dimensional structure or origin. An“isolated protein” is used to refer to a protein which is no longer inits natural environment, for example in vitro or in a recombinantbacterial or plant host cell.

The term “7-epizingiberene synthase” or “7-epizingiberene synthaseprotein” as used herein denotes a 7-epizingiberene synthase protein,i.e., the protein of the invention is capable of convertingZ,Z-farnesyl-diphosphate into 7-epizingiberene.

“Functional”, in relation to 7-epizingiberene synthase proteins (orvariants, such as orthologs or mutants, and fragments), refers to thecapability to provide insect pest resistance by modifying the expressionlevel of the 7-epizingiberene synthase-encoding gene in a plant.

The term “gene” means a DNA sequence comprising a region (transcribedregion), which is transcribed into a RNA molecule (e.g. a mRNA) in acell, operably linked to suitable regulatory regions (e.g. a promoter).A gene may thus comprise several operably linked sequences, such as apromoter, a 5′ leader sequence comprising e.g. sequences involved intranslation initiation, a (protein) coding region (cDNA or genomic DNA),introns, and a 3′non-translated sequence comprising e.g. transcriptiontermination sites.

A “chimeric gene” (or recombinant gene) refers to any gene, which is notnormally found in nature in a species, in particular a gene in which oneor more parts of the nucleic acid sequence are present that are notassociated with each other in nature. For example the promoter is notassociated in nature with part or all of the transcribed region or withanother regulatory region. The term “chimeric gene” is understood toinclude expression constructs in which a promoter or transcriptionregulatory sequence is operably linked to one or more coding sequencesor to an antisense (reverse complement of the sense strand) or invertedrepeat sequence (sense and antisense, whereby the RNA transcript formsdouble stranded RNA upon transcription).

A “3′ UTR” or “3′ non-translated sequence” (also often referred to as 3′untranslated region, or 3′end) refers to the nucleic acid sequence founddownstream of the coding sequence of a gene, which comprises for examplea transcription termination site and (in most, but not all eukaryoticmRNAs) a polyadenylation signal (such as e.g. AAUAAA or variantsthereof). After termination of transcription, the mRNA transcript may becleaved downstream of the polyadenylation signal and a poly(A) tail maybe added, which is involved in the transport of the mRNA to thecytoplasm (where translation takes place).

“Expression of a gene” refers to the process wherein a DNA region, whichis operably linked to appropriate regulatory regions, particularly apromoter, is transcribed into an RNA, which is biologically active, i.e.which is capable of being translated into a biologically active proteinor peptide (or active peptide fragment) or which is active itself (e.g.in posttranscriptional gene silencing or RNAi). An active protein incertain embodiments refers to a protein being constitutively active. Thecoding sequence is preferably in sense-orientation and encodes adesired, biologically active protein or peptide, or an active peptidefragment. In gene silencing approaches, the DNA sequence is preferablypresent in the form of an antisense DNA or an inverted repeat DNA,comprising a short sequence of the target gene in antisense or in senseand antisense orientation. “Ectopic expression” refers to expression ina tissue in which the gene is normally not expressed.

A “transcription regulatory sequence” is herein defined as a nucleicacid sequence that is capable of regulating the rate of transcription ofa (coding) sequence operably linked to the transcription regulatorysequence. A transcription regulatory sequence as herein defined willthus comprise all of the sequence elements necessary for initiation oftranscription (promoter elements), for maintaining and for regulatingtranscription, including e.g. attenuators or enhancers. Although mostlythe upstream (5′) transcription regulatory sequences of a codingsequence are referred to, regulatory sequences found downstream (3′) ofa coding sequence are also encompassed by this definition.

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one of skill in the art to act directly orindirectly to regulate the amount of transcription from the promoter. A“constitutive” promoter is a promoter that is active in most tissuesunder most physiological and developmental conditions. An “inducible”promoter is a promoter that is physiologically (e.g. by externalapplication of certain compounds) or developmentally regulated. A“tissue specific” promoter is only active in specific types of tissuesor cells. A “promoter active in plants or plant cells” refers to thegeneral capability of the promoter to drive transcription within a plantor plant cell. It does not make any implications about thespatiotemporal activity of the promoter.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter, or rather atranscription regulatory sequence, is operably linked to a codingsequence if it affects the transcription of the coding sequence.Operably linked means that the DNA sequences being linked are typicallycontiguous.

The terms “target peptide” refers to amino acid sequences which target aprotein, or protein fragment, to intracellular organelles such asplastids, preferably chloroplasts, mitochondria, or to the extracellularspace or apoplast (secretion signal peptide). A nucleic acid sequenceencoding a target peptide may be fused (in frame) to the nucleic acidsequence encoding the amino terminal end (N-terminal end) of the proteinor protein fragment, or may be used to replace a native targetingpeptide.

A “nucleic acid construct” or “vector” is herein understood to mean aman-made nucleic acid molecule resulting from the use of recombinant DNAtechnology and which is used to deliver exogenous DNA into a host cell.The vector backbone may for example be a binary or superbinary vector(see e.g. U.S. Pat. No. 5,591,616, US 2002138879 and WO95/06722), aco-integrate vector or a T-DNA vector, as known in the art and asdescribed elsewhere herein, into which a chimeric gene is integrated or,if a suitable transcription regulatory sequence is already present, onlya desired nucleic acid sequence (e.g. a coding sequence, an antisense oran inverted repeat sequence) is integrated downstream of thetranscription regulatory sequence. Vectors usually comprise furthergenetic elements to facilitate their use in molecular cloning, such ase.g. selectable markers, multiple cloning sites and the like (seebelow).

“Stringent hybridisation conditions” can be used to identify nucleotidesequences, which are substantially identical to a given nucleotidesequence. Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequences at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridises to a perfectly matchedprobe. Typically stringent conditions will be chosen in which the saltconcentration is about 0.02 molar at pH 7 and the temperature is atleast 60° C. Lowering the salt concentration and/or increasing thetemperature increases stringency. Stringent conditions for RNA-DNAhybridisations (Northern blots using a probe of e.g. 100 nt) are forexample those which include at least one wash in 0.2×SSC at 63° C. for20 min, or equivalent conditions. Stringent conditions for DNA-DNAhybridisation (Southern blots using a probe of e.g. 100 nt) are forexample those which include at least one wash (usually 2) in 0.2×SSC ata temperature of at least 50° C., usually about 55° C., for 20 min, orequivalent conditions. See also Sambrook et al. (1989) and Sambrook andRussell (2001).

“Sequence identity” and “sequence similarity” can be determined byalignment of two peptide or two nucleotide sequences using global orlocal alignment algorithms. Sequences may then be referred to as“substantially identical” or “essentially similar” when they (whenoptimally aligned by for example the programs GAP or BESTFIT usingdefault parameters) share at least a certain minimal percentage ofsequence identity (as defined below). GAP uses the Needleman and Wunschglobal alignment algorithm to align two sequences over their entirelength, maximizing the number of matches and minimises the number ofgaps. Generally, the GAP default parameters are used, with a gapcreation penalty=50 (nucleotides)/8 (proteins) and gap extensionpenalty=3 (nucleotides)/2 (proteins). For nucleotides the defaultscoring matrix used is nwsgapdna and for proteins the default scoringmatrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignments and scores for percentage sequence identity may bedetermined using computer programs, such as the GCG Wisconsin Package,Version 10.3, available from Accelrys Inc., 9685 Scranton Road, SanDiego, Calif. 92121-3752 USA, or EmbossWin version 2.10.0 (using theprogram “needle”). Alternatively percent similarity or identity may bedetermined by searching against databases, using algorithms such asFASTA, BLAST, etc. Preferably, the sequence identity refers to thesequence identity over the entire length of the sequence.

A “host cell” or a “recombinant host cell” or “transformed cell” areterms referring to a new individual cell (or organism) arising as aresult of at least one nucleic acid molecule, especially comprising achimeric gene encoding a desired protein or a nucleic acid sequencewhich upon transcription yields an antisense RNA or an inverted repeatRNA (or hairpin RNA) for silencing of a target gene/gene family, havingbeen introduced into said cell. The host cell is preferably a plant cellor a bacterial cell. The host cell may contain the nucleic acidconstruct as an extra-chromosomally (episomal) replicating molecule, ormore preferably, comprises the chimeric gene integrated in the nuclearor plastid genome of the host cell. Throughout the text the term “host”may also refer to the host plant species which a pathogen is able toinvade or infect, but this will be clear from the context. Plant speciesare classified as “host” or “non-host” species in relation to apathogen. “Non-host” species are completely immune to pathogen infectionof all races or strains of a pathogen, even under optimum conditions fordisease development. The “host” species are also referred to as the“host range” of a pathogen and are immune to certain (but not all) racesof a pathogen.

The term “selectable marker” is a term familiar to one of ordinary skillin the art and is used herein to describe any genetic entity which, whenexpressed, can be used to select for a cell or cells containing theselectable marker. Selectable marker gene products confer for exampleantibiotic resistance, or more preferably, herbicide resistance oranother selectable trait such as a phenotypic trait (e.g. a change inpigmentation) or nutritional requirements. The term “reporter” is mainlyused to refer to visible markers, such as green fluorescent protein(GFP), eGFP, luciferase, GUS and the like.

The terms “pests” and “pest” as used herein refer to “plant insectpests” or “plant pests” or “insect pests” or “plant pest species”. Suchplant insect pests include insect species that cause infestation anddamage on crop and/or ornamental plants (hosts plant species), byinfestation of the plants or plant parts. An “infestation” is thepresence of a large number of pest organisms in an area (e.g. a field orglasshouse), on the surface of a host plant or on anything that mightcontact a host plant, or in the soil. Plant insect pests includesap-sucking insect pests (see below), but also other insect pests, suchas thrips, cicada, and leaf-hoppers. The term “insect pests” as usedherein includes any herbivorous Arthropods such as mites (e.g. spidermites and others).

“Sap-sucking insect pests” include plant pests of the suborderSternorrhyncha (of the order Hemiptera, of the class Insecta), i.e.insect pests which include psyllids, whiteflies, aphids, mealybugs andscale insects and share a common property, namely the utilization ofplant sap as their food source.

“Aphids” include herein plant insect pests of the family Aphididae, suchas Aphis gossypii, A. fabae, A. glycines, A. nerii, A. nasturtii, Myzuspersicae, M. cerasi, M. ornatus, Nasonovia (e.g. N. ribisnigri),Macrosiphum, Brevicoryne and others.

“Insect vectors” are insects that are capable of carrying andtransmitting viruses, bacteria, plasmodia etc. to plants.

“Whitefly” or “whiteflies” refer to species of the genus Bemisia,especially B. tabaci and B. argentifolii (also known as biotype B of B.tabaci), and/or species of the genus Trialeurodes, especially T.vaporariorum (greenhouse whitefly) and T. abutinolea (banded wingedwhitefly).

Included herein are all biotypes, such as biotype Q and B of B. tabaci,as well as any developmental stage, such as eggs, larvae, and adults.

Throughout the application, reference is made to “7-epizingiberene”. Inthis respect, it is important to note that 7-epizingiberene is adiastereoisomer of alpha-zingiberene (Breeden and Coates, 1994,Tetrahedron, 50 (38), 11123-11132). The two molecules differ in thestereochemical configuration of one hydrogen and one methyl group:

When exposed to air, isolated 7-epizingiberene can spontaneously convertto R-curcumene. This was previously observed by Bleeker et al.(Phytochemistry. 2011 January; 72(1):68-73).

“Solanaceae” refers herein to plant genera, species, and varietiesthereof, belonging to the family Solanaceae. These include speciesbelonging to the genus Solanum (including Solanum lycopersicum, whichused to be known as Lycopersicon esculentum), Nicotiana, Capsicum,Petunia and other genera.

The term “ortholog” of a gene or protein refers herein to the homologousgene or protein found in another species, which has the same function asthe gene or protein, but (usually) diverged in sequence from the timepoint on when the species harbouring the genes diverged (i.e. the genesevolved from a common ancestor by speciation). Orthologs of the geneencoding Solanum habrochaites zingiberene synthase of the invention maythus be identified in other plant species based on both sequencecomparisons (e.g. based on percentages sequence identity over the entiresequence or over specific domains) and functional analysis.

The terms “homologous” and “heterologous” refer to the relationshipbetween a nucleic acid or amino acid sequence and its host cell ororganism, especially in the context of transgenic organisms. Ahomologous sequence is thus naturally found in the host species (e.g. atomato plant transformed with a tomato gene), while a heterologoussequence is not naturally found in the host cell (e.g. a tomato planttransformed with a sequence from potato plants). Depending on thecontext, the term “homolog” or “homologous” may alternatively refer tosequences which are descendent from a common ancestral sequence (e.g.they may be orthologs).

As used herein, the term “plant” includes plant cells, plant tissues ororgans, plant protoplasts, plant cell tissue cultures from which plantscan be regenerated, plant calli, plant cell clumps, and plant cells thatare intact in plants, or parts of plants, such as embryos, pollen,ovules, fruit (e.g. harvested tomatoes), flowers, leaves, seeds, roots,root tips and the like.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. It also encompasses the more limiting verb “to consistof”. In addition, reference to an element by the indefinite article “a”or “an” does not exclude the possibility that more than one of theelement is present, unless the context clearly requires that there beone and only one of the elements. The indefinite article “a” or “an”thus usually means “at least one”. It is further understood that, whenreferring to “sequences” herein, generally the actual physical moleculeswith a certain sequence of subunits (e.g. amino acids) are referred to.

DETAILED DESCRIPTION OF THE INVENTION Proteins and Nucleic AcidSequences

The 7-epizingiberene synthase protein of the present invention has 91%sequence identity over the entire length with a protein having GenBankentry ACJ38409.1 (710 out of 777 amino acids identical), said proteinbeing denoted as a santalene and bergamotene synthase from Solanumhabrochaites. Said protein is known to produce (+)-alpha-santalene,(−)-endo-alpha-bergomotene en (+)-endo-beta-bergamotene (Sallaud et al.,Plant Cell, vol. 21(1), 301-317, 2009 and US 2010-0138954).

In one embodiment of the invention nucleic acid sequences and amino acidsequences of 7-epizingiberene synthase proteins are provided (includingorthologs), as well as methods for isolating or identifying orthologs of7-epizingiberene synthase proteins in other plant species, such as otherSolanaceae. 7-epizingiberene synthase proteins and functional fragmentsand variants thereof, as referred to herein, are capable of producing7-epizingiberene starting from Z,Z-farnesyldiphophate (“zFPP”). Thus,such proteins, as well as functional fragments and variants thereof,have 7-epizingiberene synthase activity.

In one embodiment 7-epizingiberene synthase proteins are provided.“7-epizingiberene synthase proteins” comprise the protein depicted inSEQ ID NO:1, as well as fragments and variants thereof. Variants of7-epizingiberene synthase include, for example, proteins having at least92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%,98%, 98.5%, 99%, 99.5% or more, such as 100%, amino acid sequenceidentity over the entire length to SEQ ID NO:1. Amino acid sequenceidentity is determined by pairwise alignment using the Needleman andWunsch algorithm and GAP default parameters as defined above. Variantsalso include proteins having 7-epizingiberene activity, which have beenderived, by way of one or more amino acid substitutions, deletions orinsertions, from the polypeptide having the amino acid sequence of SEQID NO:1. Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25,20, 15 amino acid substitutions, deletions or insertions. For example,and without limitation, the following amino acids may be substituted:R10, P22, V42, K60, S90, N159, F190, 1200, V298, A304, 1310, V498, M504,S609, 1626, F646. For example, and without limitation, the followingsubstitutions may be introduced: R10Q, P22T, V42L, K60N, 590T, N159S,F190V, 1200M, V298A, A304V, 1310M, V498M, M5041, 5609T, 1626L, F646C.

Variants of 7-epizingiberene synthase can be obtained from varioussources, such as from other plant species (especially other species ofSolanaceae) or other varieties, or they can be made by de novosynthesis, mutagenesis and the like. The 7-epizingiberene synthaseproteins according to the invention may thus be isolated from naturalsources, synthesized de novo by chemical synthesis (using e.g. a peptidesynthesizer such as supplied by Applied Biosystems) or produced byrecombinant host cells by expressing the nucleic acid sequence encodingthe 7-epizingiberene synthase protein, fragment or variant. Variants andfragments are preferably functional, i.e., have 7-epizingiberenesynthase activity. When the 7-epizingiberene synthase protein of thepresent invention is not preceded by a targeting sequence as describedbelow, the 7-epizingiberene synthase protein comprising the amino acidsequence of SEQ ID NO:1 or a variant thereof as defined herein will bepreceded by a methionine residue, and the nucleic acid sequence encodingsuch protein, for example, as depicted in SEQ ID NO:2, will be precededby a start codon. In case the 7-epizingiberene synthase protein of thepresent invention is preceded by a targeting sequence, said methioninewill be encoded for within the targeting peptide.

7-epizingiberene synthase variants may comprise conservative amino acidsubstitutions within the categories basic (e.g. Arg, His, Lys), acidic(e.g. Asp, Glu), nonpolar (e.g. Ala, Val, Trp, Leu, Ile, Pro, Met, Phe,Trp) or polar (e.g. Gly, Ser, Thr, Tyr, Cys, Asn, Gln). In additionnon-conservative amino acid substitutions fall within the scope of theinvention.

The functionality of any 7-epizingiberene synthase protein, variant orfragment, can be determined using various methods. For example,transient or stable overexpression in plant cells can be used to testwhether the protein has activity, i.e. provides enhanced insect pestresistance, in planta. Functionality is preferably tested in Solanumlycopersicum. Thus, for example transient or stable expression can beused to determine whether insect pest resistance is enhanced, indicatingfunctionality.

“Fragments” of 7-epizingiberene synthase proteins and of variants of7-epizingiberene synthase proteins, as described above, comprisefragments of 100, 150, 200, 300, 400, 500, 600, 700, contiguous aminoacids or more, such as 777. Preferably, such fragments are functional inplant tissue, i.e. they are capable of conferring or enhancing insectpest resistance when produced in plant cells.

In another embodiment isolated nucleic acid sequences encoding any ofthe above proteins, variants or fragments are provided, such as cDNA,genomic DNA and RNA sequences. Due to the degeneracy of the genetic codevarious nucleic acid sequences may encode the same amino acid sequence.Any nucleic acid sequence encoding 7-epizingiberene synthase proteins orvariants thereof are herein referred to as “7-epizingiberene synthaseencoding sequences”. The nucleic acid sequences provided includenaturally occurring, artificial or synthetic nucleic acid sequences. Onesuch nucleic acid sequence encoding a 7-epizingiberene synthase proteinis provided in SEQ ID NO:2. It is understood that when sequences aredepicted as DNA sequences while RNA is referred to, the actual basesequence of the RNA molecule is identical with the difference thatthymine (T) is replace by uracil (U).

Also included are variants and fragments of 7-epizingiberene synthaseencoding nucleic acid sequences, such as nucleic acid sequenceshybridizing to 7-epizingiberene synthase encoding nucleic acid sequencesunder stringent hybridization conditions as defined. Variants of7-epizingiberene synthase encoding nucleic acid sequences also includenucleic acid sequences which have a sequence identity to SEQ ID NO:2(over the entire length) of at least 96.5%, 97%, 98%, 99%, 99.5%, 99.8%or more. It is clear that many methods can be used to identify,synthesise or isolate variants or fragments of 7-epizingiberene synthaseencoding nucleic acid sequences, such as nucleic acid hybridization, PCRtechnology, in silico analysis and nucleic acid synthesis, and the like.

The nucleic acid sequence, particularly DNA sequence, encoding the7-epizingiberene synthase proteins of this invention can be inserted inexpression vectors to produce high amounts of 7-epizingiberene synthaseproteins, as described below. For optimal expression in a host the7-epizingiberene synthase encoding DNA sequences can be codon-optimizedby adapting the codon usage to that most preferred in host (such asplant) genes. In the case of the host being a plant, codon usage may beadapted particularly to genes native to the plant genus or species ofinterest (Bennetzen & Hall, 1982, J. Biol. Chem. 257, 3026-3031; Itakuraet al., 1977 Science 198, 1056-1063.) using available codon usage tables(e.g. more adapted towards expression in cotton, soybean corn or rice).Codon usage tables for various plant species are published for exampleby Ikemura (1993, In “Plant Molecular Biology Labfax”, Croy, ed., BiosScientific Publishers Ltd.) and Nakamura et al. (2000, Nucl. Acids Res.28, 292) and in the major DNA sequence databases (e.g. EMBL atHeidelberg, Germany). Accordingly, synthetic DNA sequences can beconstructed so that the same or substantially the same proteins areproduced. Several techniques for modifying the codon usage to thatpreferred by the host cells can be found in patent and scientificliterature. The exact method of codon usage modification is not criticalfor this invention.

Small modifications to a DNA sequence such as described above can beroutinely made, i.e., by PCR-mediated mutagenesis (Ho et al., 1989, Gene77, 51-59., White et al., 1989, Trends in Genet. 5, 185-189).

“Fragments” of 7-epizingiberene synthase encoding nucleic acid sequencesinclude fragments of at least 10, 12, 15, 16, 18, 20, 30, 40, 50, 100,200, 500, 1000, 1500, 2000, 2500 or more consecutive nucleotides of SEQID NO:2, or of variants of SEQ ID NO:2. Short fragments can for examplebe used as PCR primers or hybridization probes.

In another embodiment of the invention PCR primers and/or probes andkits for detecting the 7-epizingiberene synthase encoding DNA or RNAsequences are provided. Degenerate or specific PCR primer pairs toamplify 7-epizingiberene synthase encoding DNA from samples can besynthesized based on SEQ ID NO:2 (or variants thereof) as known in theart (see Dieffenbach and Dveksler (1995) PCR Primer: A LaboratoryManual, Cold Spring Harbor Laboratory Press, and McPherson at al. (2000)PCR-Basics: From Background to Bench, First Edition, Springer Verlag,Germany). For example, any stretch of 9, 10, 11, 12, 13, 14, 15, 16, 18or more contiguous nucleotides of SEQ ID NO:2 (or the complement strand)may be used as primer or probe. Likewise, DNA fragments of SEQ ID NO:2(or variants thereof) can be used as hybridization probes. A detectionkit for 7-epizingiberene synthase encoding sequences may compriseprimers specific for 7-epizingiberene synthase encoding sequences and/orprobes specific for 7-epizingiberene synthase encoding sequences, and anassociated protocol to use the primers or probe to detect specific forzingiberene synthase encoding DNA sequences in a sample. Such adetection kit may, for example, be used to determine, whether a planthas been transformed with a specific 7-epizingiberene synthase encodinggene (or part thereof) of the invention. Because of the degeneracy ofthe genetic code, some amino acid codons can be replaced by otherswithout changing the amino acid sequence of the protein.

In yet another embodiment a method for identifying and using orthologsor alleles of the gene encoding Solanum habrochaites 7-epizingiberenesynthase (SEQ ID NO:2) is provided. The method comprises the steps of:

-   -   a) obtaining or identifying a nucleic acid sequence comprising        at least 96.5% nucleic acid identity to SEQ ID NO:2 (or a higher        percentage sequence identity, as indicated above),    -   b) using the nucleic acid sequence of a) to generate expression        and/or silencing vectors,    -   c) using one or more vectors of b) to transform a plant or plant        cell(s), preferably of the plant species from which the nucleic        acid was obtained,    -   d) analysing the capability of the transformed plant/plant        tissue to pest resistance in order to determine or verify the        gene function in planta and/or to generate transgenic plants        having enhanced insect pest resistance;    -   e) optionally, selecting those alleles or orthologs for further        use which confer enhanced pest resistance to the transgenic        plant.

Chimeric Genes, Expression Vectors, Host Cells, and RecombinantOrganisms

In one embodiment of the invention nucleic acid sequences encoding7-epizingiberene synthase proteins (including variants or fragments), asdescribed above, are used to make chimeric genes, and vectors comprisingthese for transfer of the chimeric gene into a host cell and productionof the 7-epizingiberene synthase protein(s) in host cells, such ascells, tissues, organs or organisms derived from transformed cell(s). Inan advantageous embodiment, the production of 7-epizingiberene synthaseis employed for the production of 7-epizingiberene. Vectors for theproduction of 7-epizingiberene synthase protein (or protein fragments orvariants) in plant cells are herein referred to as “expression vectors”.

Suitable host cells for expression of polypeptides such as7-epizingiberene synthase include prokaryotes, yeast, or highereukaryotic cells. Appropriate cloning and expression vectors for usewith bacterial, fungal, yeast, and mammalian cellular hosts aredescribed, for example, in Pouwels et al., Cloning vectors: A LaboratoryManual, Elsevier, N.Y., (1985). Cell-free translation systems could alsobe employed to produce the proteins of the present invention using RNAsderived from nucleic acid sequences disclosed herein. In an embodiment,said host cell (over)produces farnesyl-diphosphate (also referred to as“FPP”). In a suitable embodiment, said host cell produces oroverproduces 2Z,6Z-farnesyldiphosphate (also referred to as“Z,Z-farnesyl pyrophosphate” or “zFPP”). The skilled person is capableof overproducing the substrate of the 7-epizingiberene synthase of thepresent invention to produce 7-epizingiberene. Suitable prokaryotic hostcells include gram-negative and gram-positive organisms, for example,Escherichia coli or Bacilli. Another suitable prokaryotic host cell isAgrobacterium, in particular Agrobacterium tumefaciens.

Proteins of the present invention can also be expressed in yeast hostcells, for example from the Saccharomyces genus (e.g., Saccharomycescerevisiae). Other yeast genera, such as Pichia or Kluyveromyces, canalso be employed.

Alternatively, proteins of the present invention may be expressed inhigher eukaryotic host cells, including plant cells, fungal cells,insect cells, and mammalian, optionally non-human, cells.

One embodiment of the invention is a non-human organism modified tocomprise a nucleic acid sequence of the present invention. The non-humanorganism and/or host cell may be modified by any methods known in theart for gene transfer including, for example, the use of deliverydevices such as lipids and viral vectors, naked DNA, electroporation,chemical methods and particle-mediated gene transfer. In an advantageousembodiment, the non-human organism is a plant.

Any plant may be a suitable host, such as monocotyledonous plants ordicotyledonous plants, but most preferably the host plant belongs to thefamily Solanaceae. For example, the plant belongs to the genus Solanum(including Lycopersicon), Nicotiana, Capsicum, Petunia and other genera.The following host species may suitably be used: Tobacco (Nicotianaspecies, e.g. N. benthamiana, N. plumbaginifolia, N. tabacum, etc.),vegetable species, such as tomato (L. esculentum, syn. Solanumlycopersicum) such as e.g. cherry tomato, var. cerasiforme or curranttomato, var. pimpinellifolium) or tree tomato (S. betaceum, syn.Cyphomandra betaceae), potato (Solanum tuberosum), eggplant (Solanummelongena), pepino (Solanum muricatum), cocona (Solanum sessiliflorum)and naranjilla (Solanum quitoense), peppers (Capsicum annuum, Capsicumfrutescens, Capsicum baccatum), ornamental species (e.g. Petuniahybrida, Petunia axillaries, P. integrifolia), coffee (Coffea).

Alternatively, the plant may belong to any other family, such as to theCucurbitaceae or Gramineae. Suitable host plants include for examplemaize/corn (Zea species), wheat (Triticum species), barley (e.g. Hordeumvulgare), oat (e.g. Avena sativa), sorghum (Sorghum bicolor), rye(Secale cereale), soybean (Glycine spp, e.g. G. max), cotton (Gossypiumspecies, e.g. G. hirsutum, G. barbadense), Brassica spp. (e.g. B. napus,B. juncea, B. oleracea, B. rapa, etc), sunflower (Helianthus annus),safflower, yam, cassava, alfalfa (Medicago sativa), rice (Oryza species,e.g. O. sativa indica cultivar-group or japonica cultivar-group), foragegrasses, pearl millet (Pennisetum spp. e.g. P. glaucum), tree species(Pinus, poplar, fir, plantain, etc), tea, coffea, oil palm, coconut,vegetable species, such as pea, zucchini, beans (e.g. Phaseolusspecies), cucumber, artichoke, asparagus, broccoli, garlic, leek,lettuce, onion, radish, turnip, Brussels sprouts, carrot, cauliflower,chicory, celery, spinach, endive, fennel, beet, fleshy fruit bearingplants (grapes, peaches, plums, strawberry, mango, apple, plum, cherry,apricot, banana, blackberry, blueberry, citrus, kiwi, figs, lemon, lime,nectarines, raspberry, watermelon, orange, grapefruit, etc.), ornamentalspecies (e.g. Rose, Petunia, Chrysanthemum, Lily, Gerbera species),herbs (mint, parsley, basil, thyme, etc.), woody trees (e.g. species ofPopulus, Salix, Quercus, Eucalyptus), fibre species e.g. flax (Linumusitatissimum) and hemp (Cannabis sativa), or model organisms, such asArabidopsis thaliana.

Preferred hosts are “crop plants” or “cultivated plants”, i.e. plantspecies which is cultivated and bred by humans. A crop plant may becultivated for food or feed purposes (e.g. field crops), or forornamental purposes (e.g. production of flowers for cutting, grasses forlawns, etc.). A crop plant as defined herein also includes plants fromwhich non-food products are harvested, such as oil for fuel, plasticpolymers, pharmaceutical products, cork, fibres (such as cotton) and thelike.

The construction of chimeric genes and vectors for, preferably stable,introduction of 7-epizingiberene synthase protein-encoding nucleic acidsequences into the genome of host cells is generally known in the art.To generate a chimeric gene the nucleic acid sequence encoding a7-epizingiberene synthase protein (or variant or fragment thereof) isoperably linked to a promoter sequence, suitable for expression in thehost cells, using standard molecular biology techniques. The promotersequence may already be present in a vector so that the 7-epizingiberenesynthase nucleic acid sequence is simply inserted into the vectordownstream of the promoter sequence. The vector is then used totransform the host cells and the chimeric gene is inserted in thenuclear genome or into the plastid, mitochondrial or chloroplast genomeand expressed there using a suitable promoter (e.g., Mc Bride et al.,1995 Bio/Technology 13, 362; U.S. Pat. No. 5,693,507). In one embodimenta chimeric gene comprises a suitable promoter for expression in plantcells or microbial cells (e.g. bacteria), operably linked to a nucleicacid sequence encoding a 7-epizingiberene synthase protein according tothe invention, optionally followed by a 3′nontranslated nucleic acidsequence. The bacteria may subsequently be used for plant transformation(Agrobacterium-mediated plant transformation).

The 7-epizingiberene synthase nucleic acid sequence, preferably a7-epizingiberene synthase chimeric gene, encoding an functional7-epizingiberene synthase protein can be stably inserted in aconventional manner into the nuclear genome of a single plant cell, andthe so-transformed plant cell can be used in a conventional manner toproduce a transformed plant that has an altered phenotype due to thepresence of the 7-epizingiberene synthase protein in certain cells at acertain time. In this regard, a T-DNA vector, comprising a nucleic acidsequence encoding a zingiberene synthase protein, in Agrobacteriumtumefaciens can be used to transform the plant cell, and thereafter, atransformed plant can be regenerated from the transformed plant cellusing the procedures described, for example, in EP 0 116 718, EP 0 270822, PCT publication WO84/02913 and published European Patentapplication EP 0 242 246 and in Gould et al. (1991, Plant Physiol. 95,426-434). The construction of a T-DNA vector for Agrobacterium mediatedplant transformation is well known in the art. The T-DNA vector may beeither a binary vector as described in EP 0 120 561 and EP 0 120 515 ora co-integrate vector which can integrate into the AgrobacteriumTi-plasmid by homologous recombination, as described in EP 0 116 718.

Preferred T-DNA vectors each contain a promoter operably linked to a7-epizingiberene synthase encoding nucleic acid sequence (e.g. encodingSEQ ID NO: 2) between T-DNA border sequences, or at least located to theleft of the right border sequence. Border sequences are described inGielen et al. (1984, EMBO J 3, 835-845). Of course, other types ofvectors can be used to transform the plant cell, using procedures suchas direct gene transfer (as described, for example in EP 0 223 247),pollen mediated transformation (as described, for example in EP 0 270356 and WO85/01856), protoplast transformation as, for example,described in U.S. Pat. No. 4,684,611, plant RNA virus-mediatedtransformation (as described, for example in EP 0 067 553 and U.S. Pat.No. 4,407,956), liposome-mediated transformation (as described, forexample in U.S. Pat. No. 4,536,475), and other methods. For tomato ortobacco transformation see also An G. et al., 1986, Plant Physiol. 81:301-305; Horsch R. B. et al., 1988, In: Plant Molecular Biology ManualA5, Dordrecht, Netherlands, Kluwer Academic Publishers. pp 1-9;Koornneef M. et al., 1986, In: Nevins D. J. and R. A. Jones, eds. TomatoBiotechnology, New York, N.Y., USA, Alan R. Liss, Inc. pp 169-178). Forpotato transformation see e.g. Sherman and Bevan (1988, Plant Cell Rep.7: 13-16).

Likewise, selection and regeneration of transformed plants fromtransformed cells is well known in the art. Obviously, for differentspecies and even for different varieties or cultivars of a singlespecies, protocols are specifically adapted for regeneratingtransformants at high frequency.

Besides transformation of the nuclear genome, also transformation of theplastid genome, preferably chloroplast genome, is included in theinvention. One advantage of plastid genome transformation is that therisk of spread of the transgene(s) can be reduced. Plastid genometransformation can be carried out as known in the art, see e.g. SidorovV A et al. 1999, Plant J.19: 209-216 or Lutz K A et al. 2004, Plant J.37(6):906-13.

The resulting transformed plant can be used in a conventional plantbreeding scheme to produce more transformed plants containing thetransgene. Single copy transformants can be selected, using e.g.Southern Blot analysis or PCR based methods or the Invader® Technologyassay (Third Wave Technologies, Inc.). Alternatively, the amount of7-epizingiberene may be determined using analytical methods such asGC-MS. Transformed cells and plants can easily be distinguished fromnon-transformed ones by the presence of the chimeric gene. The sequencesof the plant DNA flanking the insertion site of the transgene can alsobe sequenced, whereby an “Event specific” detection method can bedeveloped, for routine use. See for example WO0141558, which describeselite event detection kits (such as PCR detection kits) based forexample on the integrated sequence and the flanking (genomic) sequence.

The 7-epizingiberene synthase nucleic acid sequence may be inserted in aplant cell genome so that the inserted coding sequence is downstream(i.e. 3′) of, and under the control of, a promoter which can direct theexpression in the plant cell. This is preferably accomplished byinserting the chimeric gene in the plant cell genome, particularly inthe nuclear or plastid (e.g. chloroplast) genome.

As the constitutive production of the 7-epizingiberene synthase proteinmay lead to the induction of cell death and/or may lower yield (see e.g.Rizhsky and Mittler, Plant Mol Biol, 2001 46: 313-23), it is in oneembodiment preferred to use a promoter whose activity is inducible.Examples of inducible promoters are wound-inducible promoters, such asthe MPI promoter described by Cordera et al. (1994, The Plant Journal 6,141), which is induced by wounding (such as caused by insect or physicalwounding), or the COMPTII promoter (WO0056897) or the PR1 promoterdescribed in U.S. Pat. No. 6,031,151. Alternatively the promoter may beinducible by a chemical, such as dexamethasone as described by Aoyamaand Chua (1997, Plant Journal 11: 605-612) and in U.S. Pat. No.6,063,985 or by tetracycline (TOPFREE or TOP 10 promoter, see Gatz,1997, Annu Rev Plant Physiol Plant Mol Biol. 48: 89-108 and Love et al.2000, Plant J. 21: 579-88). Other inducible promoters are for exampleinducible by a change in temperature, such as the heat shock promoterdescribed in U.S. Pat. No. 5,447,858, by anaerobic conditions (e.g. themaize ADH1S promoter), by light (U.S. Pat. No. 6,455,760), by pathogens(e.g. the gst1 promoter of EP759085 or the vst1 promoter of EP309862) orby senescence (SAG12 and SAG13, see U.S. Pat. No. 5,689,042). Obviously,there are a range of other promoters available.

In one embodiment, preferably, an insect pest inducible promoter isused, as thereby the 7-epizingiberene synthase protein (or variant orfragment) will only be produced following insect pest attack of theplant tissue. Especially, promoters of genes which are upregulatedquickly after insect pest attack are desired. Promoters inducible by aparticular plant insect pest may also be identified using known methods,such as cDNA-AFLP®.

Preferably, the promoter is inducible by a number of insect pests, i.e.it is inducible by a broad range of insect pests of the host plant. Foreach particular host plant species, a different promoter may be mostsuitable. For example, when tomato is used as a host, the promoter ispreferably induced upon at least one, but preferably more than onetomato insect pest. Especially, a promoter which is inducible by one ormore insect pests is preferred.

Detailed descriptions of plant insect pests, the disease symptoms causedby them and their life cycles can be found for each plant species. Forexample, tomato insect pests are described in “Compendium of TomatoDiseases”, Editors Jones, Jones, Stall and Zitter, ISBN 0-89054-120-5,APS Press (http:/www.shopapspress/org).

Alternatively, a host plant may comprise various 7-epizingiberenesynthase transgenes, each under control of a different pest induciblepromoter, to ensure that 7-epizingiberene synthase protein is producedfollowing attack by a variety of insect pests. For example, fortransformation of tomato, one promoter may be inducible by whitefly andone promoter may be inducible by aphids.

The word “inducible” does not necessarily require that the promoter iscompletely inactive in the absence of the inducer stimulus. A low levelnon-specific activity may be present, as long as this does not result insevere yield or quality penalty of the plants. Inducible, thus,preferably refers to an increase in activity of the promoter, resultingin an increase in transcription of the downstream zingiberene synthasecoding region following contact with the inducer.

In another embodiment constitutive promoters may be used, such as thestrong constitutive 35S promoters or enhanced 35S promoters (the “35Spromoters”) of the cauliflower mosaic virus (CaMV) of isolates CM 1841(Gardner et al., 1981, Nucleic Acids Research 9, 2871-2887), CabbB-S(Franck et al., 1980, Cell 21, 285-294) and CabbB-JI (Hull and Howell,1987, Virology 86, 482-493); the 35S promoter described by Odell et al.(1985, Nature 313, 810-812) or in U.S. Pat. No. 5,164,316, promotersfrom the ubiquitin family (e.g. the maize ubiquitin promoter ofChristensen et al., 1992, Plant Mol. Biol. 18, 675-689, EP 0 342 926,see also Cornejo et al. 1993, Plant Mol. Biol. 23, 567-581), the gos2promoter (de Pater et al., 1992 Plant J. 2, 834-844), the emu promoter(Last et al., 1990, Theor. Appl. Genet. 81, 581-588), Arabidopsis actinpromoters such as the promoter described by An et al. (1996, Plant J.10, 107.), rice actin promoters such as the promoter described by Zhanget al. (1991, The Plant Cell 3, 1155-1165) and the promoter described inU.S. Pat. No. 5,641,876 or the rice actin 2 promoter as described inW0070067; promoters of the Cassava vein mosaic virus (WO 97/48819,Verdaguer et al. 1998, Plant Mol. Biol. 37, 1055-1067), the pPLEX seriesof promoters from Subterranean Clover Stunt Virus (WO 96/06932,particularly the S7 promoter), a alcohol dehydrogenase promoter, e.g.,pAdh1S (GenBank accession numbers X04049, X00581), and the TR1′ promoterand the TR2′ promoter (the “TR1′ promoter” and “TR2′ promoter”,respectively) which drive the expression of the 1′ and 2′ genes,respectively, of the T-DNA (Velten et al., 1984, EMBO J 3, 2723-2730),the Figwort Mosaic Virus promoter described in U.S. Pat. No. 6,051,753and in EP426641, histone gene promoters, such as the Ph4a748 promoterfrom Arabidopsis (PMB 8: 179-191), or others.

Alternatively, a promoter can be utilized which is not constitutive butrather is specific for one or more tissues or organs of the plant(tissue preferred/tissue specific, including developmentally regulatedpromoters), for example leaf preferred, epidermis preferred, rootpreferred, flower tissue e.g. tapetum or anther preferred, seedpreferred, pod preferred, etc.), or trichome-specific promoters suchMTS1 and MSK1 as disclosed in WO2009082208, whereby the 7-epizingiberenesynthase gene is expressed only in cells of the specific tissue(s) ororgan(s) and/or only during a certain developmental stage. For example,the 7-epizingiberene synthase gene(s) can be selectively expressed inthe leaves of a plant by placing the coding sequence under the controlof a light-inducible promoter such as the promoter of the ribulose-1,5-bisphosphate carboxylase small subunit gene of the plant itself or ofanother plant, such as pea, as disclosed in U.S. Pat. No. 5,254,799 orArabidopsis as disclosed in U.S. Pat. No. 5,034,322.

In one embodiment the promoter of the 7-epizingiberene synthase gene ofSolanum habrochaites (wild tomato species) provided by the presentinvention is used. For example, the promoter of the 7-epizingiberenesynthase gene of S. habrochaites may be isolated and operably linked tothe coding region encoding zingiberene synthase protein of SEQ ID NO:1.The 7-epizingiberene synthase gene promoter (the upstream transcriptionregulatory region of SEQ ID NO:2) can be isolated from S. habrochaitesplants using known methods, such as TAIL-PCR (Liu et al. 1995, Genomics25(3):674-81; Liu et al. 2005, Methods Mol Biol. 286:341-8), Linker-PCR,or Inverse PCR (IPCR).

The 7-epizingiberene synthase coding sequence is preferably insertedinto the plant genome so that the coding sequence is upstream (i.e. 5′)of suitable 3′ end nontranslated region (“3′end” or 3′UTR). Suitable3′ends include those of the CaMV 35S gene (“3′ 35S”), the nopalinesynthase gene (“3′ nos”) (Depicker et al., 1982 J. Molec. Appl. Genetics1, 561-573.), the octopine synthase gene (“3′ocs”) (Gielen et al., 1984,EMBO J 3, 835-845) and the T-DNA gene 7 (“3′ gene 7”) (Velten andSchell, 1985, Nucleic Acids Research 13, 6981-6998), which act as3′-untranslated DNA sequences in transformed plant cells, and others. Inone embodiment the 3′UTR of the tomato 7-epizingiberene synthase gene ofSolanum habrochaites (wild tomato species) is used. Introduction of theT-DNA vector into Agrobacterium can be carried out using known methods,such as electroporation or triparental mating.

A 7-epizingiberene synthase encoding nucleic acid sequence canoptionally be inserted in the plant genome as a hybrid gene sequencewhereby the 7-epizingiberene synthase sequence is linked in-frame to a(U.S. Pat. No. 5,254,799; Vaeck et al., 1987, Nature 328, 33-37) geneencoding a selectable or scorable marker, such as for example the neo(or nptII) gene (EP 0 242 236) encoding kanamycin resistance, so thatthe plant expresses a fusion protein which is easily detectable.Alternatively, a 7-epizingiberene encoding nucleic acid sequence can beintroduced by means of co-transformation with a gene encoding aselectable or scorable marker, or the two genes can be present on asingle T-DNA

All or part of a 7-epizingiberene synthase nucleic acid sequence,encoding a 7-epizingiberene synthase protein (or variant or fragment),can also be used to transform microorganisms, such as bacteria (e.g.Escherichia coli, Pseudomonas, Agrobacterium, Bacillus, etc.), fungi, oralgae or insects, or to make recombinant viruses. Transformation ofbacteria, with all or part of the 7-epizingiberene synthase encodingnucleic acid sequence of this invention, incorporated in a suitablecloning vehicle, can be carried out in a conventional manner, preferablyusing conventional electroporation techniques as described in Maillon etal. (1989, FEMS Microbiol. Letters 60, 205-210.) and WO 90/06999. Forexpression in prokaryotic host cell, the codon usage of the nucleic acidsequence may be optimized accordingly (as described for plants above).Intron sequences should be removed and other adaptations for optimalexpression may be made as known.

The DNA sequence of the 7-epizingiberene synthase encoding nucleic acidsequence can be further changed in a translationally neutral manner, tomodify possibly inhibiting DNA sequences present in the gene part and/orby introducing changes to the codon usage, e.g., adapting the codonusage to that most preferred by plants, preferably the specific relevantplant genus, as described above.

In accordance with one embodiment of this invention, the7-epizingiberene synthase proteins are targeted to intracellularorganelles such as plastids, preferably chloroplasts, mitochondria, orare secreted from the cell, potentially optimizing protein stabilityand/or expression. Similarly, the protein may be targeted to vacuoles.Targeting to plastids is particularly attractive as overproduction ofsesquiterpenes in the cytosol is usually toxic to cells, whereasoverproduction of sesquiterpenes in plastids does not suffer from thisproblem. For this purpose, in one embodiment of this invention, thechimeric genes of the invention comprise a coding region encoding asignal or target peptide, linked to the 7-epizingiberene synthaseprotein coding region of the invention. The signal or target peptidemay, for example, be the natural plastid targeting peptide of said7-epizingiberene synthase, e.g., the amino acid sequence as depicted inSEQ ID NO:3 (coded for by the nucleic acid sequence of SEQ ID NO:4).Other preferred peptides to be included in the proteins of thisinvention are the transit peptides for chloroplast or other plastidtargeting, especially duplicated transit peptide regions from plantgenes whose gene product is targeted to the plastids, the optimizedtransit peptide of Capellades et al. (U.S. Pat. No. 5,635,618), thetransit peptide of ferredoxin-NADP+oxidoreductase from spinach(Oelmuller et al., 1993, Mol. Gen. Genet. 237, 261-272), the transitpeptide described in Wong et al. (1992, Plant Molec. Biol. 20, 81-93)and the targeting peptides in published PCT patent application WO00/26371. Also preferred are peptides signalling secretion of a proteinlinked to such peptide outside the cell, such as the secretion signal ofthe potato proteinase inhibitor II (Keil et al., 1986, Nucl. Acids Res.14, 5641-5650), the secretion signal of the alpha-amylase 3 gene of rice(Sutliff et al., 1991, Plant Molec. Biol. 16, 579-591) and the secretionsignal of tobacco PR1 protein (Cornelissen et al., 1986, EMBO J. 5,37-40). Particularly useful signal peptides in accordance with theinvention include the chloroplast transit peptide (e.g. Van Den Broecket al., 1985, Nature 313, 358), or the optimized chloroplast transitpeptide of U.S. Pat. No. 5,510,471 and U.S. Pat. No. 5,635,618 causingtransport of the protein to the chloroplasts, a secretory signal peptideor a peptide targeting the protein to other plastids, mitochondria, theER, or another organelle. Signal sequences for targeting tointracellular organelles or for secretion outside the plant cell or tothe cell wall are found in naturally targeted or secreted proteins,preferably those described by Klosgen et al. (1989, Mol. Gen. Genet.217, 155-161), Klosgen and Weil (1991, Mol. Gen. Genet. 225, 297-304),Neuhaus & Rogers (1998, Plant Mol. Biol. 38, 127-144), Bih et al. (1999,J. Biol. Chem. 274, 22884-22894), Morris et al. (1999, Biochem. Biophys.Res. Commun. 255, 328-333), Hesse et al. (1989, EMBO J. 8, 2453-2461),Tavladoraki et al. (1998, FEBS Lett. 426, 62-66.), Terashima et al.(1999, Appl. Microbiol. Biotechnol. 52, 516-523), Park et al. (1997, J.Biol. Chem. 272, 6876-6881), Shcherban et al. (1995, Proc. Natl. Acad.Sci USA 92, 9245-9249).

To allow secretion of the 7-epizingiberene synthase proteins to theoutside of the transformed host cell, an appropriate secretion signalpeptide may be fused to the amino terminal end (N-terminal end) of the7-epizingiberene synthase protein. Putative signal peptides can bedetected using computer based analysis, using programs such as theprogram Signal Peptide search (SignalP V3.0)(Von Heijne, Gunnar, 1986and Nielsen et al., 1996).

In one embodiment, several 7-epizingiberene synthase encoding nucleicacid sequences are co-expressed in a single host, preferably undercontrol of different promoters. Alternatively, several 7-epizingiberenesynthase protein encoding nucleic acid sequences can be present on asingle transformation vector or be co-transformed at the same time usingseparate vectors and selecting transformants comprising both chimericgenes. Similarly, one or more 7-epizingiberene synthase encoding genesmay be expressed in a single plant together with other chimeric genes,for example encoding other proteins which enhance insect pestresistance, or others.

It is understood that the different proteins can be expressed in thesame plant, or each can be expressed in a single plant and then combinedin the same plant by crossing the single plants with one another. Forexample, in hybrid seed production, each parent plant can express asingle protein. Upon crossing the parent plants to produce hybrids, bothproteins are combined in the hybrid plant.

Preferably, for selection purposes but also for weed control options,the transgenic plants of the invention are also transformed with a DNAencoding a protein conferring resistance to herbicide, such as abroad-spectrum herbicide, for example herbicides based on glufosinateammonium as active ingredient (e.g. Liberty® or BASTA; resistance isconferred by the PAT or bar gene; see EP 0 242 236 and EP 0 242 246) orglyphosate (e.g. RoundUp®; resistance is conferred by EPSPS genes, seee.g. EP 0 508 909 and EP 0 507 698). Using herbicide resistance genes(or other genes conferring a desired phenotype) as selectable markerfurther has the advantage that the introduction of antibiotic resistancegenes can be avoided.

Alternatively, other selectable marker genes may be used, such asantibiotic resistance genes. As it is generally not accepted to retainantibiotic resistance genes in the transformed host plants, these genescan be removed again following selection of the transformants. Differenttechnologies exist for removal of transgenes. One method to achieveremoval is by flanking the chimeric gene with lox sites and, followingselection, crossing the transformed plant with a CRErecombinase-expressing plant (see e.g. EP506763B1). Site specificrecombination results in excision of the marker gene. Another sitespecific recombination systems is the FLP/FRT system described inEP686191 and U.S. Pat. No. 5,527,695. Site specific recombinationsystems such as CRE/LOX and FLP/FRT may also be used for gene stackingpurposes. Further, one-component excision systems have been described,see e.g. WO9737012 or WO9500555).

The present invention encompasses a method for preparing a7-epizingiberene synthase comprising the step of culturing a host cellcomprising at least one nucleic acid molecule according to the presentinvention under conditions allowing the production of said7-epizingiberene synthase.

Also, the present invention provides a method for preparing7-epizingiberene and/or R-curcumene comprising the steps of: a)transforming a host cell with a nucleic acid molecule, chimeric gene orvector of the present invention; b) culturing said host cell underconditions permitting production of 7-epizingiberene; c) optionally,isolating the 7-epizingiberene produced in step b); and d) optionally,dehydrogenating the 7-epizingiberene to produce R-curcumene. The skilledperson will be capable of routinely selecting conditions permittingproduction of 7-epizingiberene. The host cell may have beenmetabolically engineered to produce or overproduceZ,Z-farnesyl-diphosphate (zFPP), the substrate for the 7-epizingiberenesynthase of the present invention, to produce 7-epizingiberene. Theskilled person is capable of accomplishing overproduction of thesubstrate of the 7-epizingiberene synthase of the present invention toproduce 7-epizingiberene. Similarly, a person skilled in the art will becapable of isolating the 7-epizingiberene produced using routine methodsfor isolation of volatiles.

When exposed to air, isolated 7-epizingiberene can spontaneously convertto R-curcumene. This was previously observed by Bleeker et al.(Phytochemistry. 2011 January; 72(1):68-73). Moreover, the same authorsshow that converting, for instance by controlled dehydrogenation of7-epizingiberene resulted in pure R-curcumene. The skilled person iscapable of selecting conditions permitting conversion of7-epizingiberene into R-curcumene.

zFPP, the substrate for the 7-epizingiberene synthase of the presentinvention, may be produced or overproduced by any means known in theart. For example, it may be produced naturally in the host cell ofchoice. Alternatively, a nucleic acid sequence encoding a Z,Z-farnesyldiphosphate synthase (hereinafter also referred to as “zFPS”) may beintroduced into a host cell to achieve expression of zFPP in said hostcell. Preferably, such host cell comprises a source of isopentenyldiphosphate (“IPP”) and dimethylallyl diphosphate (“DMAPP”).

An isolated or recombinant protein having Z,Z-FPS (“zFPS”) activityderived from Solanum habrochaites is described in WO 2008/142318 (hereinincorporated by reference) and can further be found in the GenBankaccession no. ACJ38408.1. As used in the context of the presentinvention, the term “Z,Z-farnesyl diphosphate synthase” or “zFPS”denotes a protein having an amino acid sequence as depicted in SEQ IDNO:6 or a variant thereof having at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acidsequence of SEQ ID NO:6, preferably over the full length.

The present invention therefore also relates to a method for producing7-epizingiberene from zFPP in a host cell, comprising:

-   -   a) introducing into said host cell a first nucleic acid sequence        encoding a zFPS as described herein and a second nucleic acid        sequence encoding the 7-epizingiberene synthase of the present        invention;    -   b) culturing the transformed cell in suitable conditions for the        expression of said first and said second nucleic acid sequences;        and,    -   c) optionally, collecting the zFPP and/or the 7-epizingiberene        contained in said cell and/or in the culture medium.

The first nucleic acid sequence and second nucleic acid sequence may bepresent in a single vector or may be present in separate vectors.

Transformed plant cells/plants/seeds and uses of the nucleic acidsequence and proteins according to the invention

In the following part the use of the 7-epizingiberene synthase-encodingnucleic acid sequences according to the invention to generate transgenicplant cells, plants, plant seeds, etc. and any derivatives/progenythereof, with an enhanced insect pest resistance phenotype is described.

A transgenic plant with enhanced insect pest resistance can be generatedby transforming a plant host cell with a nucleic acid sequence encodingat least one 7-epizingiberene synthase protein under the control of asuitable promoter, as described above, and regenerating a transgenicplant from said cell.

Preferred promoters are promoters which are insect pest inducible, asdescribed above.

Preferably, the transgenic plants of the invention comprise enhancedinsect pest resistance against one or more insect pests, especially.Thus, for example transgenic tomato or potato plants comprise enhancedresistance to at least one, or more, of the insect species listed above.

“Insect pest resistance” or “increased/enhanced insect pest resistance”is used herein to refer to an enhanced ability of plants harbouring thenucleotide sequence of the present invention (compared to wild type orcontrol plants not harbouring the nucleotide sequence of the presentinvention) to withstand the attack of one or more plant insect pests, orin other words, it refers to a significant reduction in disease symptomsin plants harbouring the nucleotide sequence of the present inventioncompared to plants not harbouring the nucleotide sequence of the presentinvention (or empty-vector transformed) controls. Insect pest resistanceor enhanced insect pest resistance may be determined using a variety ofmethods. Often disease symptoms are scored visually (either in bioassaysor in the field) by assessing the disease symptoms at one or more timepoints after infestation or contact with an insect pest. Alternativemethods include methods whereby the insect pest is detected andoptionally quantified. A (transgenic) plant may thus show enhancedinsect pest resistance if the amount or number of insect pests detectedin/on the tissue is significantly less compared to controls, or if theinsect pest spread is significantly slower than in controls. Ultimately,a significant increase in average yield of plants harbouring thenucleotide sequence of the present invention (e.g. at least 1%, 2%, 5%,10% or more) compared to controls, when grown under equivalent insectpest pressure (preferably in the field) provides an indirect measurementof enhanced insect pest resistance.

Thus, a plurality of plants harbouring the nucleotide sequence of thepresent invention, for example, transgenic plants, expressing the7-epizingiberene synthase protein of the invention show enhanced insectpest resistance if they show a significant reduction of diseasesymptoms, compared to the plants not harbouring the nucleotide sequenceof the present invention. Obviously, statistical analysis is required todetermine whether significant difference exists. Preferably, one or moredisease symptoms are on average at least 2%, 5%, 10%, 15%, 20%, 30%,40%, 50%, or even 100% lower in 7-epizingiberene synthase encodingnucleic acid sequence-harbouring plants than in the control plants. Asthe disease assay is different for every host-insect pest combination,no specific protocol can be provided, but the skilled person knows howto determine whether plants harbouring the nucleotide sequence of thepresent invention show significantly enhanced disease resistance to oneor more insect pests. Bioassays as known in the art for each plant-pestcombination can be used to compare resistance of transgenic plants tosuitable controls.

Generally, the role of 7-epizingiberene produced by the protein encodedby the amino acid sequence of SEQ ID NO:1 in resistance (toxicity and/orrepellance) to insect pests will be determined through the use of choiceand no-choice experiments. In particular, a choice test will beperformed. In a choice test different life stages (e.g. larvae oradults) will be allowed to choose between (transgenic) plants thatproduce 7-epizingiberene (through expression of a nucleic acid sequenceencoding the amino acid sequence of SEQ ID NO:1) and non-transgenic (orempty vector) plants. This test will determine the repellent activity of7-epizingiberene produced by the protein of SEQ ID NO:1.

A no-choice test will also be performed to determine the toxic effectsof 7-epizingiberene produced through the expression of a nucleic acidsequence encoding the amino acid sequence of SEQ ID NO:1. In theseexperiments, insect pest species are forced to eat from (transgenic)plants that produce 7-epizingiberene (through expression of a nucleicacid sequence encoding the amino acid sequence of SEQ ID NO:1) andnon-transgenic (or empty vector) plants. Subsequently, the insectperformance (e.g. growth, development or fitness) will be determined asa measure of toxicity.

It is also an embodiment to generate transgenic plants which expressseveral 7-epizingiberene synthase proteins, preferably under the controlof different promoters, such as different pest inducible promoters.

The disease resistance phenotype can be fine-tuned by expressing asuitable amount of 7-epizingiberene synthase protein at a suitable timeand location. Such fine-tuning may be done by determining the mostappropriate promoter for a particular host-pest combination and also byselecting transgenic “events” which show the desired expression level. Atoo low level of 7-epizingiberene synthase protein or too slow inductionof 7-epizingiberene synthase protein production following insect pestattack may be insufficient to enhance disease resistance levels. On theother hand, a too high protein level or expression at times andlocations devoid of insect pest attack, may result in agronomicallyundesired phenotypes and yield penalties. However, the skilled personcan easily generate plants having enhanced disease resistance, but whichat the same time are agronomical acceptable.

Plants harbouring the nucleotide sequence of the present inventionexpressing desired levels of the 7-epizingiberene synthase protein areselected by e.g. analysing copy number (Southern blot analysis), mRNAtranscript levels, by analysing the presence and level of7-epizingiberene synthase protein in various tissues (e.g. SDS-PAGE;ELISA assays, etc), or by determining the amount of 7-epizingiberene,using analytical methods such as GC-MS. For regulatory reasons,preferably single copy transformants are selected and the sequencesflanking the site of insertion of the chimeric gene is analysed,preferably sequenced to characterize the “event”. High or moderate7-epizingiberene synthase-encoding nucleic acid sequence expressingtransgenic events are selected for further crossing/backcrossing/selfinguntil a high performing elite event with a stable 7-epizingiberenesynthase-encoding nucleic acid sequence transgene is obtained.

Transformants expressing one or more 7-epizingiberene synthase genesaccording to the invention may also comprise other transgenes, such asother genes conferring disease resistance or conferring tolerance toother biotic and/or abiotic stresses. To obtain such plants with“stacked” transgenes, other transgenes may either be introgressed intothe 7-epizingiberene synthase-encoding nucleic acid sequencetransformants, or 7-epizingiberene synthase-encoding nucleic acidsequence transformants may be transformed subsequently with one or moreother genes, or alternatively several chimeric genes may be used totransform a plant line or variety. For example, several chimeric genesmay be present on a single vector, or may be present on differentvectors which are co-transformed.

In one embodiment the following genes are combined with one or more7-epizingiberene synthase genes according to the invention: knowndisease resistance genes, especially genes conferring enhancedresistance to pathogens, virus resistance genes, abiotic stressresistance genes (e.g. drought tolerance, salt tolerance, heat- or coldtolerance, etc.), herbicide resistance genes, and the like. The stackedtransformants may thus have an even broader biotic and/or abiotic stresstolerance, to pathogen resistance, nematode resistance, salinity, coldstress, heat stress, water stress, etc. Also, 7-epizingiberenesynthase-encoding nucleic acid sequence silencing approaches may becombined with 7-epizingiberene synthase-encoding nucleic acid sequenceexpression approaches in a single plant. For example, 7-epizingiberenesynthase-encoding nucleic acid sequence overexpression in roots ortubers may confer or enhance root or tuber resistance to soil pests.

It is also possible to introduce or introgress the 7-epizingiberenesynthase gene into a plant breeding line which already has a certainlevel of insect pest resistance. For durability of insect pestresistance in the field, it may be desirable to stack several diseaseresistance mechanisms in a plant, preferably whereby the resistancesources have different underlying molecular mechanisms.

Whole plants, seeds, cells, tissues and progeny (such as F1 hybrids, F2seeds/plants, etc.) of any of the transformed plants described above areencompassed herein and can be identified by the presence of thetransgene in the DNA, for example by PCR analysis using total genomicDNA as template and using zingiberene synthase-encoding nucleic acidsequence specific PCR primer pairs. Also “event specific” PCR diagnosticmethods can be developed, where the PCR primers are based on the plantDNA flanking the inserted chimeric gene, see U.S. Pat. No. 6,563,026.Similarly, event specific AFLP fingerprints or RFLP fingerprints may bedeveloped which identify the transgenic plant or any plant, seed, tissueor cells derived there from.

It is understood that the transgenic plants according to the inventionpreferably do not show non-desired phenotypes, such as yield reduction,enhanced susceptibility to diseases or undesired architectural changes(dwarfing, deformations) etc. and that, if such phenotypes are seen inthe primary transformants, these can be removed by normal breeding andselection methods (crossing/backcrossing/selfing, etc.). Any of thetransgenic plants described herein may be homozygous or hemizygous forthe transgene.

The present invention also relates to a Solanum lycopersicum orLycopersicon esculentum plant, plant cell, seed or fruit, comprising anucleotide sequence encoding the amino acid sequence of SEQ ID NO:1 oran amino acid sequence comprising at least 92% amino acid sequenceidentity to the amino acid sequence of SEQ ID NO:1 over the entirelength. Wild type Solanum lycopersicum does not produce detectableamounts of 7-epi-zingiberene. Using the nucleotide sequence of thepresent invention, it is possible to prepare a transgenic ornon-transgenic Solanum lycopersicum plant, plant cell, seed or fruithaving enhanced insect pest resistance. Preferably, said Solanumlycopersicum plant, plant cell, seed or fruit further comprises anucleic acid sequence encoding a Z,Z-farnesyl diphosphate synthase.

Sequences referred to

SEQ ID NO 1: amino acid sequence of the Solanumhabrochaites plastid zingiberene synthase protein.CSHSTPSSMNGFEDARDRIRESFGKVELSPSSYDTAWVAMVPSKHSLNEPCFPQCLDWIIENQREDGSWGLNPSHPLLLKDSLSSTLACLLALTKWRVGDEQIKRGLGFIETQSWAIDNKDQISPLGFEIIFPSMIKSAEKLNLNLAINKRDSTIKRALQNEFTRNIEYMSEGFGELCDWKEIIKLHQRQNGSLFDSPATTAAALIYHQHDKKCYEYLNSILQQHKNWVPTMYPTKIHSLLCLVDTLQNLGVHRHFKSEIKKALDEIYRLWQQKNEEIFSNVTHCAMAFRLLRISYYDVSSDELAEFVDEEHFFATSGKYTSHVEILELHKASQLAIDHEKDDILDKINNWTRTFMEQKLLNNGFIDRMSKKEVELALRNFYIISDLAENRRYIKSYEENNFKILKAAYRSPNINNKDLFIFSIRDFELCQAQHQEELQQLKRWFEDCRLDQLGLSEQFISASYLCAIPIVPGPELSDARLVYAKYVMLLTIVDDHFESFASTDECLNIIELVERWDDYASVGYKSERVKVLFSMFYKSIEEIATIAEIKQGRSVKNHLINLWLKVMKLMLMERVEWCSGKTIPRIEEYLYVSSITFGSRLIPLTTQYFIGIKISKDLLESDEIYGLCNFTGIVLRLLNDLQDSKREQKEGSINLVTLLMKSISEEEAIMKMKEILEMKRRELFKMVLVQKKGSQLPQLCKEIFWRTCKWAHFTYSQTDRYRFPEEMENHIDEVFYKPLNHSEQ ID NO 2: nucleic acid sequence of the Solanumhabrochaites zingiberene synthase gene (coding sequence only).TGCAGCCACAGTACCCCTTCATCAATGAATGGTTTCGAAGATGCAAGGGATAGAATAAGGGAAAGTTTTGGGAAAGTAGAGTTATCTCCTTCTTCCTATGACACAGCATGGGTAGCTATGGTCCCTTCAAAACATTCACTAAATGAGCCATGTTTTCCACAATGTTTGGATTGGATTATTGAAAATCAAAGAGAAGATGGATCTTGGGGACTAAACCCTAGCCATCCATTGCTTCTTAAGGACTCACTTTCTTCCACTCTTGCATGTTTGCTTGCACTAACCAAATGGAGAGTTGGAGATGAGCAAATCAAAAGAGGCCTTGGCTTTATTGAAACCCAGAGTTGGGCAATTGATAACAAGGATCAAATTTCACCTCTAGGATTTGAAATTATATTTCCCAGTATGATCAAGTCTGCAGAAAAACTAAACTTAAATCTAGCAATTAACAAAAGAGATTCAACAATTAAAAGAGCATTACAGAATGAGTTCACGAGGAATATTGAATATATGAGTGAAGGATTTGGTGAATTATGTGATTGGAAGGAAATAATAAAGTTACATCAAAGGCAAAATGGTTCATTATTTGATTCACCAGCCACTACTGCAGCTGCCTTGATTTACCATCAGCATGATAAAAAATGCTATGAATATCTTAATTCAATCTTGCAACAACACAAAAATTGGGTTCCCACTATGTATCCAACAAAGATACATTCATTGCTTTGCTTGGTTGATACACTTCAAAATCTTGGAGTACATCGGCATTTTAAATCAGAAATAAAGAAAGCCCTAGATGAAATATACAGGCTATGGCAACAAAAGAATGAAGAAATTTTCTCAAATGTCACCCATTGTGCTATGGCTTTTCGACTTCTAAGGATAAGCTACTATGATGTCTCCTCAGATGAACTAGCAGAATTTGTGGATGAAGAACATTTCTTTGCAACAAGTGGGAAATATACAAGTCATGTTGAAATTCTTGAACTCCACAAAGCATCACAATTGGCTATTGATCATGAGAAAGATGACATTTTGGATAAGATTAACAATTGGACAAGAACATTTATGGAGCAAAAACTCTTAAACAATGGCTTCATAGATAGGATGTCAAAAAAGGAGGTGGAACTTGCTTTGAGGAATTTTTATATCATATCTGATCTAGCAGAAAATAGAAGATATATAAAGTCATACGAAGAGAACAATTTTAAAATCTTAAAAGCAGCTTATAGGTCACCTAACATTAACAATAAGGACTTGTTTATATTTTCAATACGCGACTTTGAATTATGCCAAGCTCAACACCAAGAAGAACTTCAACAACTCAAGAGGTGGTTTGAAGATTGTAGATTGGACCAACTCGGACTTTCGGAACAATTTATATCTGCTAGTTACTTATGTGCTATTCCTATTGTCCCCGGGCCTGAATTATCCGATGCTCGTCTCGTGTACGCGAAATACGTCATGCTCTTGACTATTGTCGATGATCATTTCGAGAGTTTTGCATCTACAGATGAATGTCTCAACATCATTGAATTAGTAGAAAGGTGGGATGACTATGCAAGTGTAGGTTATAAATCTGAGAGGGTTAAAGTTTTATTTTCAATGTTTTACAAATCAATAGAGGAGATTGCAACAATTGCTGAAATTAAACAAGGACGATCTGTCAAAAATCACCTTATTAATTTGTGGCTTAAAGTGATGAAGTTGATGTTGATGGAACGAGTAGAGTGGTGTTCTGGCAAGACAATACCAAGAATAGAAGAGTATTTGTATGTTAGTTCTATAACATTTGGTTCAAGATTGATTCCTCTCACAACACAATATTTTATTGGAATAAAAATATCCAAAGATCTTTTAGAAAGTGATGAAATTTATGGTTTATGCAATTTTACCGGTATAGTCTTGAGGCTCCTCAATGATTTACAAGATTCCAAGAGAGAACAAAAGGAGGGCTCAATAAATTTAGTCACATTACTAATGAAAAGTATCTCTGAGGAAGAAGCTATAATGAAGATGAAGGAAATCTTGGAAATGAAAAGAAGAGAGTTATTTAAAATGGTTTTAGTTCAAAAAAAGGGAAGCCAATTGCCTCAATTATGCAAAGAAATATTTTGGAGGACATGCAAATGGGCTCATTTCACTTATTCACAAACTGATAGATATAGATTTCCAGAGGAAATGGAGAATCACATTGATGAAGTCTTTTACAAACCACTCAATCATTAASEQ ID NO: 3. Amino acid sequence of the plastidtargeting sequence of the zingiberene synthase of the inventionMIVGYRSTIITLSHPKLGNGKTISSNAIFRRSCRVRSEQ ID NO: 4. Nucleic acid sequence of the plastidtargeting sequence of the zingiberene synthase of the inventionATGATAGTTGGCTATAGAAGCACAATCATAACCCTTTCTCATCCTAAGCTAGGCAATGGGAAAACAATTTCATCCAATGCAATTTTCCGGAGATCATGTA GAGTAAGASEQ ID NO: 5. Nucleic acid sequence of zFPS fromS. habrochaites PI127826GCTCGTGGACTCAACAAGATTTCATGCTCACTCAGCTTACAAACCGAAAAACTTTGTTATGAGGATAATGATAATGATCTTGATGAAGAACTTATGCCTAAACACATTGCTTTGATAATGGATGGTAATAGGAGATGGGCAAAGGATAAGGGTTTAGACGTATCCGAAGGTCACAAACATCTCTTTCCAAAATTAAAAGAGATTTGTGACATTTCTTCTAAATTGGGAATACAAGTTATCACTGCTTTTGCATTCTCTACTGAAAATTGGAAACGAGCCAAGGGGGAGGTTGATTTCTTGATGCAAATGTTCGAAGAACTCTATGATGAGTTTTCGAGGTCTGGAGTAAGAGTGTCTATTATTGGTTGTAAAACCGACCTCCCAATGACATTACAAAAATGCATAGCATTAACAGAAGAGACTACAAAGGGAAACAAAGGACTTCACCTTGTGATTGCACTAAACTATGGTGGATATTATGACATATTGCAAGCAACAAAAAGCATTGTTAATAAAGCAATGAATGGTTTATTAGATGTAGAAAATATCAACAAGAATTTATTTGATCAAGAACTTGAAAGCAAGTGTCCAAATCCTGATTTACTTATAAGGACAGGAGGTGTTCAAAGAGTTAGTAACTTTTTGTTGTGGCAATTGGCTTATACTGAATTTTACTTCACCAAAACATTGTTTCCTGATTTTGGAGAGGAAGATCTTAAAGAGGCAATAATAAACTTTCAACAAAGGCATAGACGTTTTGGTGGACACACATATTGASEQ ID NO: 6. Amino acid sequence of zFPS from S. habrochaites PI127826ARGLNKISCSLSLQTEKLCYEDNDNDLDEELMPKHIALIMDGNRRWAKDKGLDVSEGHKHLFPKLKEICDISSKLGIQVITAFAFSTENWKRAKGEVDFLMQMFEELYDEFSRSGVRVSIIGCKTDLPMTLQKCIALTEETTKGNKGLHLVIALNYGGYYDILQATKSIVNKAMNGLLDVENINKNLFDQELESKCPNPDLLIRTGGVQRVSNFLLWQLAYTEFYFTKTLFPDFGEEDLKEAIINFQQRH RRFGGHTYSEQ ID NO: 7. Signal peptide of zFPS from S. habrochaites PI127826MSSLVLQCWKLSSPSLILQQNTSISMGAFKGIHKLQIPNSPLTVSSEQ ID NO: 8 Nucleic acid sequence of the signalpeptide of zFPS from S. habrochaites PI127826ATGAGTTCTTTGGTTCTTCAATGTTGGAAATTATCATCTCCATCTCTGATTTTACAACAAAATACATCAATATCCATGGGTGCATTCAAAGGTATTCATAAACTTCAAATCCCAAATTCACCTCTGACAGTGTCT

FIGURES

FIG. 1 shows Gas Chromatography Mass Spectrometry (GCMS) results ofproduction of 7-epizingiberene using zFPP as precursor by E. colitransformed with the nucleotide sequence encoding 7-epizingiberenesynthase. 7-epizingiberene was identified by its MS ion massfingerprint, retention time and Kovats index.

FIGS. 2A and 2B show a mass spectra of 7-epizingiberene produced byexpression of the nucleotide sequence encoding 7-epizingiberene synthase(FIG. 2A) in E. coli and 7-epizingiberene produced by S. habrochaitesPI127826 trichomes (FIG. 2B).

FIG. 3A shows the determination of the enantiomer of ShZIS

Enantioselective gas chromatography on the cyclodextrin coated columnallowed identification of the different zingiberene stereoisomers (AstecCHIRALDEX™ B-DM column, Supelco). From top to bottom:

S. habrochaites=positive control for 7-epizingiberene;F2 ShxSI=the F2 produces 7-epizingiberene with zFPP as precursorgingeroil=positive control for alpha-zingiberene.ShZIS+ginger oil=ShZIS with zFPP produces 7-epizingiberene and gingeroil contains alpha-zingiberene

FIG. 3A indicates that ShZIS synthesizes 7-epizingiberene when providedwith zFPP. Moreover, it shows evidence that F2 plants used in thebioassays also produce 7-epizingiberene.

FIG. 3B shows the determination of the enantiomer of the zingibereneproduced by ShZIS. Enantioselective gas chromatography on thecyclodextrin coated column allowed identification of the differentzingiberene stereoisomers (Astec CHIRALDEX™ B-DM column, Supelco). Fromtop to bottom:

S. habrochaites=positive control for 7-epizingiberene;αZIS+FPP=alpha-zingiberene synthase provided with FPP yieldsα-zingiberene;αZIS+FPP & ShZIS+zFPP=alpha-zingiberene synthase provided with FPPyields alpha-zingiberene and ShZIS synthesizes 7-epizingiberene whenprovided with zFPP.Ginger oil=positive control for alpha-zingiberene.ShZIS+zFPP=the protein ShZIS, when supplied with zFPP as a precursorproduces 7-epizingiberene.

The figure indicates that ShZIS synthesizes 7-epizingiberene whenprovided with zFPP. Lemon basil zingiberene synthase (ObZIS; Iijima etal., 2004) is a bona-fide alpha-zingiberene synthase when provided withFPP.

FIGS. 4A and 4B show production of 7-epizingiberene in transgenic S.lycopersicum plants, when both zFPS and ShZIS are expressed undertrichome specific promoters. FIG. 4A. The production of 7-epizingibereneas measure by GCMS. Depicted are the terpenoid profiles of untransformedcontrol (S. lyc) plants, S. lycopersicum plants transformed only withzFPS, and S. lycopersicum plants transformed with both zFPS and ShZISunder trichome-specific promoters. 7-epizingiberene was only produced inplants transformed with zFPS and ShZIS (both under trichome-specificpromoters). FIG. 4B. Enantioselective gas chromatography on thecyclodextrin coated column proved that zingiberene production in S.lycopersicum plants transformed with zFPS and ShZIS (S. lycopersicumzFPS-ZIS in the figure), like in wild S. habrochaites, is7-epizingiberene.

FIG. 5 shows the concentration of zingiberene (ng terpenes per mg leafFW) in three different genotypes. An interspecies cross between S.lycopersicum and S. habrochaites was performed and F2 lines were testedfor the production of zingiberene. Cuttings were made of zingibereneproducing F2 lines, S. lycopersicum C32 (Moneymaker) and of S.habrochaites (PI127826). F2 and S. habrochaites (PI127826) plants showedsimilar amounts of 7-epizingiberene, no 7-epizingiberene was detectablein S. lycopersicum C32.

FIG. 6A shows the percentage of dead B. tabaci adults (mortality) inthree different genotypes. Clip cage experiments were performed oncuttings of F2 lines, S. lycopersicum C32 (Moneymaker) and of S.habrochaites (PI127826). Compared to S. habrochaites and F2 plants, thepercentage dead adults after 5 days was significantly lower on S.lycopersicum (FIG. 4A; One-way ANOVA, LSD; p<0.05 for both comparisons).

FIG. 6B shows the total number of eggs deposited by whitefly adults infive days. Clip cage experiments were performed on cuttings of F2 lines,S. lycopersicum C32 and of S. habrochaites (PI127826). The number ofeggs deposited by female B. tabaci adults was significantly higher on S.lycopersicum C32 compared to either F2 or S. habrochaites plants (FIG.4B; One-way ANOVA, LSD; p<0.05 for both comparisons).

FIG. 7A shows 7-epizingiberene levels F2 plants expressed as[zingiberene] (ng mg-1 FW leaf).

FIG. 7B shows Colorado Potato Beetle (CPB) neonate larvae survival in abio-assay (24 hrs feeding).

FIG. 7C shows feeding damage by CPB—24 hrs of feeding.

FIG. 7D shows feeding damage by CPB—24 hrs of feeding—damage isclassified as arbitrary units (pixels).

FIG. 8 demonstrates the preference of the Greenhouse whitefly(Trialeurodes vaporariorum) preference in a choice-assay for low7-epizingiberene producing plants (line F2-45) over high7-epizingiberene (line F2-40) plants.

FIG. 9 shows performance of the potato/tomato aphid (Macrosiphumeuphorbiae) in a no-choice-assay between low 7-epizingiberene producingplants (line F2-45) and high 7-epizingiberene producing (line F2-40)plants.

FIG. 10A demonstrates oviposition of Tuta absoluta. Tuta absoluta mothswere released in a cage on F2 plants that produced a range of7-epizingiberene. These plants have arisen from an interspecies crossbetween S. lycopersicum (C32) and S. habrochaites (PI127826). The numberof eggs per tomato genotype was determined after 5 days.

FIG. 10B shows production of 7-epizingiberene in these F2 plants arisenfrom an interspecies cross between S. lycopersicum (C32) and S.habrochaites (PI127826).

FIG. 11A demonstrates Spider mite (T. urticae) fecundity on S.lycopersicum (C32), 7-epi-zingiberene producing transgenic S.lycopersicum (line 2) and S. habrochaites (PI127826).

FIG. 11B shows Spider mite (T. urticae) survival on S. lycopersicum(C32), 7-epi-zingiberene producing transgenic S. lycopersicum (line 2)and S. habrochaites (PI127826).

FIG. 11C displays Spider mite (T. evansi) fecundity on S. lycopersicum(C32), 7-epi-zingiberene producing transgenic S. lycopersicum (line 2)and S. habrochaites (PI127826).

FIG. 11D demonstrates Spider mite (T. evansi) survival on S.lycopersicum (C32), 7-epi-zingiberene producing transgenic S.lycopersicum (line 2) and S. habrochaites (PI127826).

The following non-limiting Examples illustrate the different embodimentsof the invention. Unless stated otherwise in the Examples, allrecombinant DNA techniques are carried out according to standardprotocols as described in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual,Third Edition, Cold Spring Harbor Laboratory Press, NY; and in Volumes 1and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology,Current Protocols, USA. Standard materials and methods for plantmolecular work are described in Plant Molecular Biology Labfax (1993) byR.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK)and Blackwell Scientific Publications, UK.

All references recited in the present disclosure are herein incorporatedby reference.

EXAMPLES Example 1 E. coli Expression Assay to Determine Production of7-Epizingiberene

The full length gene (comprising SEQ ID NO:4 5′ of SEQ ID NO:2 (SEQ IDNO:4-SEQ ID NO:2)) was cloned into the pGEX-KG expression vector (Guanand Dixon 1991). Constructs were transformed to C41 (DE3) E. coli cells(Dumon-Seignovert et al., 2004). As a control, empty pGEX-KG vector wastransformed. A culture was grown to an OD₆₀₀ of 0.5-0.6 at 37° C. andplaced at 4° C. for 30 min. Protein expression was induced with 1 mMisopropyl β-D-1-thiogalactopyranoside (IPTG). After 16 hours incubationat 16° C., cells were harvested by centrifugation. The supernatant wasremoved and the pellet was resuspended in assay buffer (25 mM HEPES, pH7.2, 10 mM MgCl₂, 10% (v/v) glycerol) with added lysozyme (1 mg mL⁻¹)and proteinase inhibitors and incubated on ice for 30 minutes andsubsequently sonicated. The lysate was centrifuged and the supernatantwas stored at −80° C. Activity assays were performed in 500 μL 50 mMHEPES, pH7.2, 100 mM KCl, 7.5 mM MgCl₂, 20 μM MgCl₂, 5% (v/v) glycerol,5 mM DTT with 50 μL protein and 2 mM cis-FPP(2Z-6Z-farnesyl diphosphate)as substrate. Enzyme products were analyzed by GC-MS with Solid PhaseMicro Extraction fiber (SPME). Terpene products were identified usingstandards and comparing ion spectra, retention time and Kovats Index(see FIGS. 1 and 2).

Example 2 Determining the Enantiomer of Zingiberene Produced by theVarious Genes/Proteins

Activity assays were performed in 20 mL glass vials in a total volume of500 μL 50 mM HEPES, pH7.2, 100 mM KCl, 7.5 mM MgCl₂, 20 μM MgCl₂, 5%(v/v) glycerol, 5 mM DTT with 50 μL protein and either 2 mM cis-FPP(2Z-6Z-farnesyl diphosphate), trans-FPP (E-E-farnesyl diphosphate), GPP(geranyl diphosphate), NDP (neryl diphosphate), or GGPP (geranylgeranyldiphosphate) as a substrate (Echelon Biosciences Incorporated, Salt LakeCity, USA). Vials were closed with a teflon lined crimp cap immediatelyand incubated under moderate shaking for 1 hour at 30° C.

Enzyme products were sampled with a Solid Phase Micro Extraction fiber(SPME) for 10 minutes after the vial had been agitated and heated to 50°C. The fiber was desorbed for 1 minute in an Optic injector port (ATASGL Int. Zoeterwoude, NL) which was kept at 220° C. For liquid injection1-3 μL of sample in hexane was injected.

In order to separate alpha-zingiberene from 7-epizingiberene the AstecCHIRALDEX™ B-DM column (30m×0.25 mm×0.12 μm film thickness; Supelco) wasselected. The column was placed in an 6890 N gas chromatograph (Agilent,Amstelveen, NI). The programme was initially set to 115° C. for 3minutes, increased to 140° C. by 4° C. min⁻¹ where it was kept for anadditional minute after which the temperature slowly increased (2° C.min⁻¹) to 166° C. by where it was kept for 5 minutes prior to a rapidincrease with 40° C. min⁻¹ to 220° C. Helium was used as a carrier gas.Mass spectra were generated with the ion source set to −70V at 200° C.and collected with a Time-of-Flight MS (Leco, Pegasus III, St. Joseph,Mich., USA) at 1850 V, with an acquisition rate of 20 scans per second.

Run time 1800 sec; Initial injector temperature: 220° C.; Final injectortemperature: 220° C.; Transfer column flow: 1.5 mL min⁻¹; Transfer time:120 sec; Initial column flow: 1 mL min⁻¹; Final column flow: 1 mL min⁻¹;Split flow: 25 mL min⁻¹

More columns specifics can be obtained via the supplier or via:http://www.sigmaaldrich.com/catalog/ProductDetail.do?D7=0&N5=SEARCH_CONCAT_PNO|BRAND_KEY&N4=66023AST|SUPELCO&N25=0&QS=ON&F=SPEC

Results

Enantioselective gas chromatography on the cyclodextrin coated columnallowed identification of the different zingiberene stereoisomers in S.habrochaites and ginger, previously inseparable by our GC-MS analysis.By NMR it was determined that S. habrochaites PI127826 produces7-epizingiberene, whereas gingeroil contains alpha-zingiberene (Bleekeret al., 2011). Extracts from S. habrochaites and gingeroil were used aspositive controls to study the enantiomer status of zingiberenesynthesized by ObZIS (sweet lemon basil zingiberene synthase; Iijima etal., 2004) and ShZIS. Analysis (both liquid and SPME) indicated that theenzymes synthesize different stereoisomers. ShZIS is responsible for theproduction of 7-epizingiberene (similar to the enantiomer found in S.habrochaites), whereas ObZIS is a bona-fide alpha-zingiberene synthase(FIG. 3A,B).

FIG. 3A:

Liquid injection of samples in hexane. S. habrochaites leaf wash:standard for 7-epizingiberene (RT:844). Ginger oil: standard foralpha-zingiberene (RT: 851) and S-curcumene (RT:829). Mix leafwash andgingeroil: S-curcumene (RT:829), 7 epizingiberene (RT:844) andalpha-zingiberene (RT:851). Hexane overlay of E. coli C41 (DE3)transformed with pGEX:ZIS2 incubated with zFPP: 7-epizingiberene(RT:844). This experiment shows separation of 7-epizingiberene andalpha-zingiberene (previously identified with NMR in Bleeker et al.,2011) on the chiral column and proves that heterologously expressed7-epizingiberene synthase (ShZIS) is responsible for 7-epizingiberene inPI127826. It also shows that the F2 plant produces 7-epizingiberene.

FIG. 3B:

SPME: S. habrochaites leaf material as standard for 7-epizingiberene(RT: 850) and R-curcumene (RT:841). Gingeroil as standard foralpha-zingiberene (RT:856) and S-curcumene (RT:835). Sweet lemon basilObZIS (lijima, R. Davidovich-Rikanati, E. Fridman, D. R. Gang, E. Bar,E. Lewinsohn, and E. Pichersky (2004). The Biochemical and MolecularBasis for the Divergent Patterns in the Biosynthesis of Terpenes andPhenylpropenes in the Peltate Glands of Three Cultivars of Basil. PlantPhysiology 136; 3724-3736.) heterologously expressed and provided withE-E-FPP made alpha-zingiberene (RT:856). PI127826 ShZIS heterologouslyexpressed and provided with Z-Z-FPP made 7-epizingiberene (RT:850).Mixed ObZIS and ShZIS showed both peaks. This experiments shows thatShZIS makes a different zingiberene stereoisomer than known plantzingiberene synthase ObZIS.

Example 3 Development of Transgenic S. lycopersicum Plants TomatoCotyledon Explant Transformation Experiments

Tomato (S. lycopersicum) line C32 was used for transformations withAgrobacterium tumefaciens (GV3101). The tomato transformation protocolhas been described in Koornneef et al (1986) (Koornneef, Maarten,Jongsma, Maarten, Weide, Rob, Zabel, Pim, and Hille, Jacques. (1986);Transformation of tomato. In: Tomato Biotechnology, Donald Nevins andRichard Jones, eds. Alan Liss Inc., New York, USA, pg. 169-178.) and inKoornneef et al (1987) (Koornneef, M., Hanhart, C. J., and Martinelli,L. (1987); A genetic analysis of cell culture traits in tomato. Theor.Appl. Genet. 74: 633-641). Trichome-specific targeting was ensured usingMKS1 (methylketone synthase 1; Fridman et al., 2005 (Fridman E, Wang J,Iijima Y, Froehlich J E, Gang D R, Ohlrogge J, Pichersky E (2005).Metabolic, genomic, and biochemical analyses of glandular trichomes fromthe wild tomato species Lycopersicon hirsutum identify a key enzyme inthe biosynthesis of methylketones. Plant Cell 17: 1252-1267)) and MTS1(monoterpene synthase 1; WO2009082208) from S. habrochaites and S.lycopersicum, respectively. For co-transformation, Agrobacteriumcarrying a binary vector with pMKS1:zFPS and pMTS1:ShZIS were dilutedcultures are mixed in a ratio of 1:1. The remainder of the describedprotocol has been unchanged. When tomato shoots appeared, they wereharvested and rooted on solid MS20 medium containing 1 mg L⁻¹ IBA, 200mg L⁻¹ cefotaxime, 200 mg L⁻¹ vancomycin, and 100 mg L⁻¹ kanamycin.

Genomic DNA was isolated from transgenic plants and PCR was performed onTO plants to confirm successful insertion of the plasmids. Leaf materialof TO plants was harvested and analyzed by GC-MS as described above.

Results:

Bleeker et al. (2009) previously have shown that 7-epizingiberene isproduced by S. habrochaites PI127826. The gene responsible for theproduction of 7-epizingiberene, called ShZIS, was isolated from S.habrochaites PI127826. Transgenic plants were produced byAgrobacterium-mediated transformation of S. lycopersicum C32. Whereas no7-epizingiberene was formed in S. lycopersicum (C32) control plants orplants transformed with MKS1:zFPS only, 7-epizingiberene was present intransgenic S. lycopersicum plants with zFPS and ShZIS inserted intotheir genome (FIG. 4).

Example 5 Effect of the Expression of the 7-EpizingibereneSynthase-Encoding Nucleotide Sequence on Insect Pest ResistanceMethodology Bioassays:

An interspecies cross between S. lycopersicum and S. habrochaites wasperformed and F2 lines were transferred to the greenhouse at theUniversity of Amsterdam. The F2 plants were tested for their productionof 7-epizingiberene. Cuttings were made of 7-epizingiberene producing F2lines, S. lycopersicum C32 (moneymaker) and of S. habrochaites(PI127826). Both a parental line of the initial cross.

Bioassay B. tabaci (Whitefly)

Two cuttings of genotypes PI127826 and C32 and the respective F2sreceived 4 clip cages, each of which contained 20 adult B. tabaci(biotype Q) initially collected in Almeria (Spain) and rearedcontinuously on cucumber under laboratory conditions (see Bleeker etal., 2009—Plant Physiol.). After 5 days, the total number and percentageof dead flies and total nr of eggs (combined abaxial and adaxial side ofleaves) was determined.

In addition, leaf material of same leaflet was used to determine theterpene concentrations.

F2 plants have 7-epizingiberene levels comparable to S. habrochaites(PI127826). 7-epizingiberene was not detected in S. lycopersicum (C32).

Results Bioassay B. tabaci:

Cuttings of an F2 plant and S. habrochaites (PI127826) showed similaramounts of 7-epizingiberene. No 7-epizingiberene could be detected in S.lycopersicum C32 (FIG. 5). Moreover, increased resistance to whiteflieswas observed in cuttings of the F2 plant and S. habrochaites (PI127826)(FIG. 6A,B). Compared to S. habrochaites and F2 plants, the percentagedead adults after 5 days was significantly lower on S. lycopersicum(FIG. 6A; One-way ANOVA, LSD; p<0.05 for both comparisons). Moreover,the number of eggs deposited by female B. tabaci adults wassignificantly higher on S. lycopersicum C32 compared to either F2 or S.habrochaites plants (FIG. 6B; One-way ANOVA, LSD; p<0.05 for bothcomparisons). Both the mortality and oviposition characteristics showthat 7-epizingiberene produced by plants enhances resistance towhiteflies.

Bio-Assay Colorado Potato Beetle (CPB)

Larvae of the CPB, Leptinotarsa decemlineata (order: Coleoptera) werereared on potato (cultivar Bintje). A no-choice assay with performed for24 hours. CPB larvae (neonates) were allowed to feed on leaf discs (1.2cm diameter) of F2 plants arisen from an interspecies cross between S.lycopersicum (C32) and S. habrochaites (PI127826).

F2 plant 40 shows high levels of zingiberene (similar to S.habrochaites), whereas F2 plant 45 only produced minute levels ofzingiberene. Leaf discs from both genotypes were placed on wetted filterpaper in a Petri dish and one larvae was allowed to feed for 24 hours(10 biological replicates per plant genotype). Subsequently, larvaesurvival and feeding damage were assessed.

Result Bio-Assay CPB

7-epizingiberene levels were measured using the method described above.The F2 plant 40 shows high concentration of 7-epizingiberene, whereas F2plant 45 produces only minute levels of 7-epizingiberene (concentrationat the detection limit; FIG. 7A).

Larvael survival after 24 hours of feeding was significantly differenton the two genotypes. Only 20% of the larvae survived on the high7-epizingiberene producing F2-40 plant. In contrast, most larvaesurvived (70%) and were feeding from the low-producing plant (F2-45;FIG. 7B). Feeding damage was assessed by scanning the leaf discs.Significantly more damage was observed on plants with low7-epizingiberene (plant F2-45; FIG. 7C). Moreover, damage due to CPBfeeding was quantified by using ImageJ. The analysis determines thenumber of pixels (arbitrary units) of scanned leaf discs. Damage wasdetermined as the number of pixels for undamaged leaf discs compared tothat of CPB damaged leaf discs. FIG. 7D indicates that significantlymore damage was observed on leaf discs of plant F2-45, compared to F2-40(high 7-epizingiberene production).

Bio-Assay Trialeurodes vaporariorum (Greenhouse Whitefly)

Trialeurodes vaporariorum (order: Hemiptera) were reared on tomato (S.lycopersicum). A choice assay with performed for 24 hours. Adults werereleased in a cage with two F2 plants arisen from an interspecies crossbetween S. lycopersicum (C32) and S. habrochaites (PI127826).Subsequently, adult settling preference was determined on leaves of thefollowing F2 plants (10 leaves per plant). F2 plant 40 showed highlevels of 7-epizingiberene (similar to S. habrochaites), whereas F2plant 45 only produced minute levels of 7-epi-zingiberene (FIG. 7A).

Results Bio-Assay Trialeurodes vaporariorum

Greenhouse whitefly preference was different on the two genotypes (FIG.8). Compared to the high 7-epizingiberene producing plants (F2-40),twice as many greenhouse whitefly adults settled on the low7-epizingiberene producing plants (F2-45).

Bio-Assay Macrosiphum euphorbiae (Potato/Tomato Aphid)

Macrosiphum euphorbiae (order: Hemiptera) were reared on tomato (S.lycopersicum). A no-choice assay was performed for 48 hours. One adultaphid was placed in a clip-cage on either of two F2 plants arisen froman interspecies cross between S. lycopersicum (C32) and S. habrochaites(PI127826). Subsequently, aphid performance (survival and number ofoffspring) was determined on the following F2 plants (3 clip-cages perplant; 6 plants per genotype). F2 plant 40 shows high levels of7-epizingiberene (similar to S. habrochaites), whereas F2 plant 45 onlyproduced minute levels of 7-epizingiberene (FIG. 7A).

Results Bio-Assay Macrosiphum euphorbiae

Aphid performance was different on the two genotypes (FIG. 9). Comparedto the high 7-epizingiberene producing plants (F2-40), aphids performedbetter in terms of survival and number of offspring produced on low7-epizingiberene producing plants (F2-45).

Bio-Assay Tuta absoluta

Tuta absoluta (order: Lepidoptera) were reared on tomato (S.lycopersicum). A no-choice assay was performed for 7 days. 5 adults wereallowed to oviposit their eggs on S. lycopersicum (C32) plants and on F2plants arisen from an interspecies cross between S. lycopersicum (C32)and S. habrochaites (PI127826). After 7 days, Tuta abosoluta oviposition(number of eggs deposited) was determined on the abaxial and adaxialside of six leaves per plant genotype. F2 plants were characterized for7-epizingiberene content after the assay and Tuta abosoluta oviposition(number of eggs deposited) was correlated to the content of7-epizingiberene.

Results Bio-Assay Tuta absoluta

Oviposition by Tuta absoluta females was significantly reduced on F2plants producing 7-epizingiberene (FIG. 10 a). FIG. 10 b indicates the7-epizingiberene concentration in the F2 plants tested for Tuta absolutaovipostition. Oviposition was negatively correlated with7-epizingiberene content (combination of FIGS. 10A and 10B).

Bio-Assay Spider Mites

Spider mites, like insects, belong to the arthropods but are a differentclass of organisms. The effect of 7-epizingiberene was tested on twospider mite species, Tetranychus urticae and T. evansi. Both arthropodspecies were reared on common garden bean. A 4-day no-choice assay wasperformed with synchronized populations of T. urticae and T. evansi.Mites were place on leaf discs of susceptible control plants (S.lycopersicum), resistant S. habrochaites PI127826 plants and on7-epizingiberene producing transgenic S. lycopersicum plants (line 2).Subsequently, mite survival and fecundity (number of eggs/mite) wasassessed. Transgenic plants were made as described above. In short,plants were co-transformed with two constructs to produce7-epizingiberene in glandular trichomes of S. lycopersicum (pMKS1:zFPSand pMTS1:ShZIS). In this experiment one transgenic line was used (line2).

Results Bio-Assay Spider Mites

Mite fecundity was reduced by the production of 7-epi-zingiberene intransgenic S. lycopersicum plants. Compared to S. lycopersicum,transgenic plants that produced 7-epi-zingiberene showed reduced mitesurvival (both species). Moreover, FIGS. 11A and 11C indicate a strongreduction of mite fecundity (eggs/mite) for both T. urticae and T.evansi, 81% and 54% reduction, respectively.

Overall survival was also impacted for both spider mite species. FIGS.11B and 11D indicate that the percentage of dead spider mites was higheron transgenic plants producing 7-epizingiberene compared tonon-7-epizingiberene-producing S. lycopersicum plants (S. lyc 32).

Example 5 7-Epi-Zingiberene Production in Various Plants

Arabidopsis thaliana, Nicotiana tabacum, Cucumis melo, Lactuca sativa,Glycine max, and Gossypium hirsutum are co-transformed with ShzFPS(additional zFPP precursor) and ShZIS (encoding 7-epizingiberenesynthase). 7-epi-zingiberene production in the leaves of co-transformedplants is compared to 7-epi-zingiberene production in mock-transformedplants of the same species. Arabidopsis thaliana, Nicotiana tabacum,Cucumis melo, Lactuca sativa, Glycine max, and Gossypium hirsutum arecapable of producing 7-epi-zingiberene.

1. A Solanum lycopersicum plant, plant cell or tissue culture, seed, orfruit, comprising a nucleotide sequence encoding the amino acid sequenceof SEQ ID NO:1 or an amino acid sequence comprising at least 92% aminoacid sequence identity to the amino acid sequence of SEQ ID NO:1.
 2. TheSolanum lycopersicum plant, plant cell or tissue culture, seed, or fruitaccording to claim 1, which has enhanced 7-epizingiberene productioncompared to a wild-type Solanum lycopersicum plant.
 3. The Solanumlycopersicum plant, plant cell or tissue culture, seed, or fruitaccording to claim 1, which has enhanced insect pest resistance comparedto a wild-type Solanum lycopersicum plant.
 4. The Solanum lycopersicumplant, plant cell or tissue culture, seed, or fruit according to claim1, which has enhanced resistance to whiteflies compared to a wild-typeSolanum lycopersicum plant.
 5. The Solanum lycopersicum plant, plantcell or tissue culture, seed, or fruit according to claim 1, comprisinga nucleotide sequence encoding an amino acid sequence comprising atleast 95% amino acid sequence identity to the amino acid sequence of SEQID NO:1.
 6. The Solanum lycopersicum plant, plant cell or tissueculture, seed, or fruit according to claim 1, comprising a nucleotidesequence encoding an amino acid sequence comprising at least 99% aminoacid sequence identity to the amino acid sequence of SEQ ID NO:1.
 7. TheSolanum lycopersicum plant, plant cell or tissue culture, seed, or fruitaccording to claim 1, further comprising a nucleic acid sequenceencoding the amino acid sequence of SEQ ID NO:6 or an amino acidsequence comprising at least 80% amino acid sequence identity to theamino acid sequence of SEQ ID NO:6.
 8. The Solanum lycopersicum plant,plant cell or tissue culture, seed, or fruit according to claim 7, whichhas enhanced 7-epizingiberene production compared to a wild-type Solanumlycopersicum plant.
 9. The Solanum lycopersicum plant, plant cell ortissue culture, seed, or fruit according to claim 7, which has enhancedinsect pest resistance compared to a wild-type Solanum lycopersicumplant.
 10. The Solanum lycopersicum plant, plant cell or tissue culture,seed, or fruit according to claim 7, which has enhanced resistance towhiteflies compared to a wild-type Solanum lycopersicum plant.
 11. TheSolanum lycopersicum plant, plant cell or tissue culture, seed, or fruitaccording to claim 1, further comprising a nucleic acid sequenceencoding an amino acid sequence comprising at least 90% amino acidsequence identity to the amino acid sequence of SEQ ID NO:6.
 12. TheSolanum lycopersicum plant, plant cell or tissue culture, seed, or fruitaccording to claim 1, further comprising a nucleic acid sequenceencoding an amino acid sequence comprising at least 95% amino acidsequence identity to the amino acid sequence of SEQ ID NO:6.
 13. TheSolanum lycopersicum plant, plant cell or tissue culture, seed, or fruitaccording to claim 1, which is a Solanum lycopersicum seed.
 14. A plantgrown from the Solanum lycopersicum seed of claim
 13. 15. The Solanumlycopersicum plant, plant cell or tissue culture, seed, or fruitaccording to claim 1, which is a Solanum lycopersicum plant cell ortissue culture.
 16. A plant regenerated from the Solanum lycopersicumplant cell or tissue culture of claim
 15. 17. The Solanum lycopersicumplant, plant cell or tissue culture, seed, or fruit according to claim1, which is a Solanum lycopersicum plant.
 18. The Solanum lycopersicumplant according to claim 17, wherein the nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:1 or an amino acid sequencecomprising at least 92% amino acid sequence identity to the amino acidsequence of SEQ ID NO:1 is introgressed into the Solanum lycopersicumplant.